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.

CRISPR/Cas9-mediated knockin of IRES-tdTomato at Ins2 locus reveals no RFP-positive cells in mouse islets.

Using the CRISPR/Cas9 genomic editing technology, we constructed a transgenic mouse model to express specific fluorescent protein in pancreatic ß cells, which harbor tdTomato exogenous gene downstream of the Ins2 promoter in C57BL/6 J mice. The Ins2-specific single-guide RNA-targeted exon2 was designed for the CRISPR/Cas9 system and Donor vector was constructed at the same time. Then Cas9, sgRNA, and Donor vector were microinjected in vitro into the mouse zygotes that were implanted into pseudo-pregnant mice. We obtained homozygotes through mating heterozygotes, and verified the knockin effect through genotype identification, in vivo imaging, and frozen section. Six F0 mice and stable inherited Ins2-IRES-tdTomato F1 were obtained. Genome sequencing results showed that the knockin group had no change in the Ins2 exon compared with the control group, while only the base sequence of tdTomato was added and no base mutation occurred. However, in vivo imaging and frozen section did not observe the expression of red fluorescent protein (RFP), and the protein expression of knockin gene tdTomato was negative. As a result, the expressions of tdTomato protein and fluorescence intensity were low and the detection threshold was not reached. In the CRISP/Cas9 technique, the exogenous fragment of IRES connection would affect the transcription level of the preceding gene, which in turn would lead to low-level expression of the downstream gene and affect the effect of gene insertion.

OVEREXPRESSION OF PTEN GENE INCREASES INS2 GENE MRNA EXPRESSION, NOT INS1 GENE MRNA EXPRESSION, IN INSULINOMA CELL LINE RIN-5F.

Objective: One functional neuroendocrine tumor that causes hypoglycemia due to inappropriately high insulin production is an insulinoma. In rats, two genes coding for insulin, insulin 1 (Ins1) and insulin 2 (Ins2) are found on chromosome 1. Ins1 was produced from an Ins2 transcript, and it was inserted into the genome via an RNA-mediated duplication-transposition event, according to some structural feature analyses. Methods: In this study, the author has looked at how overexpression of the PTEN gene in the insulinoma cell line Rin-5F affects the expression of the insulin genes, Ins 1 and Ins 2. Results: In the insulinoma cell line, overexpression of the PTEN gene boosts Ins2 gene mRNA expression but not Ins1 gene mRNA expression. It has been reported that PTEN upregulates insulin signaling by increasing insulin receptor substrate (IRS)-2 mRNA levels. Also, PTEN has been reported to be secreted in exosomes and thereafter, into extracellular space. Conclusions: The present study suggested that overexpression of PTEN might induce the increasing Ins 2 gene expression, one of the phosphorylated genes against the IRS-2 through the insulin/IGF-1 receptor. Our knowledge of the molecular pathways of PTEN relating the synthesis of insulin has been increased by the present study.

OVEREXPRESSION OF PDX-1 GENE INCREASES INS1 GENE mRNA EXPRESSION, NOT INS2 GENE mRNA EXPRESSION, IN INSULINOMA CELL LINE RIN-5F.

Objective: Insulinoma is one of the functional neuroendocrine tumors, which induce hypoglycemia caused by inadequate high secretion of insulin. In rats, two genes coding for insulin, insulin 1 (Ins1) and insulin 2 (Ins2) are found on chromosome 1. Some structural feature studies have shown that Ins1 was generated from a transcript of Ins2 and was inserted into the genome by an RNA-mediated duplication-transposition event. Methods: In this study, the author has investigated how the expression of insulin genes, Ins 1 and Ins2, are altered by Pdx-1 gene overexpression in the insulinoma cell line, Rin-5F. Results: Overexpression of the Pdx-1 gene increases Ins1 gene mRNA expression, not Ins2 gene mRNA expression, in the insulinoma cell line. Thus, levels of the rat insulin 1 and insulin 2 peptides may be changed under specific conditions. Conclusions: This is the first report that Ins1, but not Ins2, is significantly increased by Pdx-1 gene overexpression in the insulinoma cell line. This could indicate more research and analysis of insulinoma tumorigenesis and Pdx-1 gene expression.

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.

Altered bone microarchitecture in a type 1 diabetes mouse model Ins2Akita.

Type 1 diabetes mellitus (T1DM) has been associated to several cartilage and bone alterations including growth retardation, increased fracture risk, and bone loss. To determine the effect of long term diabetes on bone we used adult and aging Ins2 Akita mice that developed T1DM around 3-4 weeks after birth. Both Ins2 Akita and wild-type (WT) mice were analyzed at 4, 6, and 12 months to assess bone parameters such as femur length, growth plate thickness and number of mature and preapoptotic chondrocytes. In addition, bone microarchitecture of the cortical and trabecular regions was measured by microcomputed tomography and gene expression of Adamst-5, Col2, Igf1, Runx2, Acp5, and Oc was quantified by quantitative real-time polymerase chain reaction. Ins2 Akita mice showed a decreased longitudinal growth of the femur that was related to decreased growth plate thickness, lower number of chondrocytes and to a higher number of preapoptotic cells. These changes were associated with higher expression of Adamst-5, suggesting higher cartilage degradation, and with low expression levels of Igf1 and Col2 that reflect the decreased growth ability of diabetic mice. Ins2 Akita bone morphology was characterized by low cortical bone area (Ct.Ar) but higher trabecular bone volume (BV/TV) and expression analysis showed a downregulation of bone markers Acp5, Oc, and Runx2. Serum levels of insulin and leptin were found to be reduced at all-time points Ins2 Akita . We suggest that Ins2 Akita mice bone phenotype is caused by lower bone formation and even lower bone resorption due to insulin deficiency and to a possible relation with low leptin signaling.

Effect of diabetes and hyaluronidase on the retinal endothelial glycocalyx in mice.

We sought to investigate the effects of diabetes and hyaluronidase on the thickness of the endothelial glycocalyx layer in the mouse retina. In our study, the retinal circulation of diabetic Ins2(Akita) mice and their nondiabetic littermates were observed via intravital microscopy. The endothelial glycocalyx thickness was determined from the infusion of two fluorescently labeled plasma markers, one of which was a high molecular weight rhodamine dextran (MW = 155,000) excluded from the glycocalyx, and the other a more permeable low molecular weight sodium fluorescein (MW = 376). In nondiabetic C57BL/6 mice, the glycocalyx thickness also was evaluated prior to and following infusion of hyaluronidase, an enzyme that can degrade hyaluronic acid on the endothelial surface. A leakage index was used to evaluate the influence of hyaluronidase on the transport of the fluorescent tracers from the plasma into the surrounding tissue, and plasma samples were obtained to measure levels of circulating hyaluronic acid. Both diabetes and hyaluronidase infusion significantly reduced the thickness of the glycocalyx in retinal arterioles (but not in venules), and hyaluronidase increased retinal microvascular leakage of both fluorescent tracers into the surrounding tissue. However, only hyaluronidase infusion (not diabetes) increased circulating plasma levels of hyaluronic acid. In summary, our findings demonstrate that diabetes and hyaluronidase reduce the thickness of the retinal endothelial glycocalyx, in which hyaluronic acid may play a significant role in barrier function.

Long-Term Diabetic Microenvironment Augments the Decay Rate of Capsaicin-Induced Currents in Mouse Dorsal Root Ganglion Neurons.

Individuals with end-stage diabetic peripheral neuropathy present with decreased pain sensation. Transient receptor potential vanilloid type 1 (TRPV1) is implicated in pain signaling and resides on sensory dorsal root ganglion (DRG) neurons. We investigated the expression and functional activity of TRPV1 in DRG neurons of the Ins2+/Akita mouse at 9 months of diabetes using immunohistochemistry, live single cell calcium imaging, and whole-cell patch-clamp electrophysiology. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence assay was used to determine the level of Reactive Oxygen Species (ROS) in DRGs. Although TRPV1 expressing neuron percentage was increased in Ins2+/Akita DRGs at 9 months of diabetes compared to control, capsaicin-induced Ca2+ influx was smaller in isolated Ins2+/Akita DRG neurons, indicating impaired TRPV1 function. Consistently, capsaicin-induced Ca2+ influx was decreased in control DRG neurons cultured in the presence of 25 mM glucose for seven days versus those cultured with 5.5 mM glucose. The high glucose environment increased cytoplasmic ROS accumulation in cultured DRG neurons. Patch-clamp recordings revealed that capsaicin-activated currents decayed faster in isolated Ins2+/Akita DRG neurons as compared to those in control neurons. We propose that in poorly controlled diabetes, the accelerated rate of capsaicin-sensitive TRPV1 current decay in DRG neurons decreases overall TRPV1 activity and contributes to peripheral neuropathy.

Antagonising Wnt/ß-catenin signalling ameliorates lens-capsulotomy-induced retinal degeneration in a mouse model of diabetes.

AIMS/HYPOTHESIS: Cataract surgery in diabetic individuals worsens pre-existing retinopathy and triggers the development of diabetic ocular complications, although the underlying cellular and molecular pathophysiology remains elusive. We hypothesise that lens surgery may exaggerate pre-existing retinal inflammation in diabetes, which may accelerate neurovascular degeneration in diabetic eyes. METHODS: Male heterozygous Ins2Akita mice (3 months of age) and C57BL/6 J age-matched siblings received either lens capsulotomy (to mimic human cataract surgery) or corneal incision (sham surgery) in the right eye. At different days post surgery, inflammation in anterior/posterior ocular tissues was assessed by immunohistochemistry and proinflammatory gene expression in the retina by quantitative PCR (qPCR). Degenerative changes in the retina were evaluated by electroretinography, in vivo examination of retinal thickness (using spectral domain optical coherence tomography [SD-OCT]) and morphometric analysis of retinal neurons. The therapeutic benefit of neutralising Wnt/ß-catenin signalling following lens capsulotomy was evaluated by intravitreal administration of monoclonal antibody against the co-receptor low-density lipoprotein receptor-related protein 6 (LRP6) (Mab2F1; 5 µg/µl in each eye). RESULTS: Lens capsulotomy triggered the early onset of retinal neurodegeneration in Ins2Akita mice, evidenced by abnormal scotopic a- and b-wave responses, reduced retinal thickness and degeneration of outer/inner retinal neurons. Diabetic Ins2Akita mice also had a higher number of infiltrating ionised calcium-binding adapter molecule 1 (IBA1)/CD68+ cells in the anterior/posterior ocular tissues and increased retinal expression of inflammatory mediators (chemokine [C-C motif] ligand 2 [CCL2] and IL-1ß). The expression of ß-catenin was significantly increased in the inner nuclear layer, ganglion cells and infiltrating immune cells in Ins2Akita mice receiving capsulotomy. Neutralisation of Wnt/ß-catenin signalling by Mab2F1 ameliorated ocular inflammation and prevented capsulotomy-induced retinal degeneration in the Ins2Akita mouse model of diabetes. CONCLUSIONS/INTERPRETATION: Targeting the canonical Wnt/ß-catenin signalling pathway may provide a novel approach for the postoperative management of diabetic individuals needing cataract surgery.

Evaluation of Notch3 Deficiency in Diabetes-Induced Pericyte Loss in the Retina.

Loss of vascular pericytes has long been associated with the onset of diabetic retinopathy; however, mechanisms contributing to pericyte dropout are not understood. Notch3 has been implicated in pericyte stability and survival, and linked to vascular integrity. Notch3 mutant mice exhibit progressive loss of retinal pericytes. Given that diabetic retinopathy is associated with pericyte loss, we sought to determine whether perturbation of Notch3 signaling contributes to diabetes-induced pericyte dropout and capillary degeneration. We utilized a pericyte-expressed LacZ transgene (XlacZ4) to examine pericyte loss in retinas of a type I diabetic mouse model (Ins2Akita) and Notch3-deficient mice. Notch3 null animals showed a dramatic loss of the LacZ marker by 8 weeks of age, while Ins2Akita diabetic and Notch3 heterozygous mice exhibited a much slower and subtler loss of LacZ. Although combined Notch3 heterozygosity in Ins2Akita diabetic animals did not show further deficits, the trypsin digest method revealed that Notch3 haploinsufficiency increased the formation of acellular capillaries in diabetic mice. Our data further indicate that Notch signaling is blunted in diabetic retinas and in cells exposed to hyperglycemia. These results are the first to demonstrate an association between Notch3 signaling, pericyte loss, and diabetic retinopathy.

Loss-of-function mutation of serine racemase attenuates retinal ganglion cell loss in diabetic mice.

Consistent results suggest the promoting roles of serine racemase (SR)/D-serine in retinal neurodegeneration in diabetic retinopathy (DR). However, the direct evidence connecting SR deficiency with retinal neuroprotection in genetic model of diabetes mellitus has not been reported. In this investigation, we explore the effect of absence of functional SR on the degeneration of retinal ganglion cells (RGCs) with a diabetic murine model, Ins2Akita mice. We established a murine strain with double mutation, termed Ins2Akita-Srr, by mating heterozygous Ins2Akita mice with homozygous Srrochre269 mice. Ins2Akita retained less RGC in posterior, middle, and peripheral retinae than the counterpart from non-diabetic sibling mice at the age of five or seven months. Ins2Akita-Srr mice retained more RGC in middle and peripheral--but not in posterior-- retinae than the counterpart from Ins2Akita sibling mice at the age of five months. By contrast, at the age of seven months, Ins2Akita-Srr mice contained more RGC in peripheral, middle, and posterior retinae than the counterpart from Ins2Akita. RGCs were identified with retrograde labeling in vivo or with immunolabeling against a RGC-specific transcription factor, Brn3a, in retinal flat mounts. Correspondingly, the aqueous humor of Ins2Akita-Srr contained less amount of D-serine than sibling Ins2Akita mice. Thus, SR deficiency significantly prevented RGC loss in diabetic mice. We conclude that D-serine is a critical factor in the degeneration of RGC in DR. Targeting SR expression or activity may be a strategy for ameliorating RGC loss in DR.

Preserving expression of Pdx1 improves ß-cell failure in diabetic mice.

Pdx1, a ß-cell-specific transcription factor, has been shown to play a crucial role in maintaining ß-cell function through transactivation of ß-cell-related genes. In addition, it has been reported that the expression levels of Pdx1 are compromised under diabetic conditions in human and rodent models. We therefore aimed to clarify the possible beneficial role of Pdx1 against ß-cell failure and generated the transgenic mouse that expressed Pdx1 conditionally and specifically in ß cells (ßPdx1) and crossed these mice with Ins2Akita diabetic mice. Whereas Pdx1 mRNA levels were reduced in Ins2Akita mice compared with their non-diabetic littermates, the mRNA levels of Pdx1 were significantly recovered in the islets of ßPdx1; Ins2Akita mice. The ßPdx1; Ins2Akita mice exhibited significantly improved glucose tolerance, compared with control Ins2Akita littermates, accompanied by increased insulin secretion after glucose loading. Furthermore, histological examination demonstrated that ßPdx1; Ins2Akita mice had improved localization of SLC2A2 (GLUT2), and quantitative RT-PCR showed the recovered expression of Mafa and Gck mRNAs in the islets of ßPdx1; Ins2Akita mice. These findings suggest that the sustained expression of Pdx1 improves ß-cell failure in Ins2Akita mice, at least partially through the preserving expression of ß-cell-specific genes as well as improved localization of GLUT2.

Homology modeling of Homo sapiens lipoic acid synthase: Substrate docking and insights on its binding mode.

Lipoic acid synthase (LIAS) is an iron-sulfur cluster mitochondrial enzyme which catalyzes the final step in the de novo pathway for the biosynthesis of lipoic acid, a potent antioxidant. Recently there has been significant interest in its role in metabolic diseases and its deficiency in LIAS expression has been linked to conditions such as diabetes, atherosclerosis and neonatal-onset epilepsy, suggesting a strong inverse correlation between LIAS reduction and disease status. In this study we use a bioinformatics approach to predict its structure, which would be helpful to understanding its role. A homology model for LIAS protein was generated using X-ray crystallographic structure of Thermosynechococcus elongatus BP-1 (PDB ID: 4U0P). The predicted structure has 93% of the residues in the most favour region of Ramachandran plot. The active site of LIAS protein was mapped and docked with S-Adenosyl Methionine (SAM) using GOLD software. The LIAS-SAM complex was further refined using molecular dynamics simulation within the subsite 1 and subsite 3 of the active site. To the best of our knowledge, this is the first study to report a reliable homology model of LIAS protein. This study will facilitate a better understanding mode of action of the enzyme-substrate complex for future studies in designing drugs that can target LIAS protein.

Methylation of insulin DNA in response to proinflammatory cytokines during the progression of autoimmune diabetes in NOD mice.

AIMS/HYPOTHESIS: Type 1 diabetes is caused by the immunological destruction of pancreatic beta cells. Preclinical and clinical data indicate that there are changes in beta cell function at different stages of the disease, but the fate of beta cells has not been closely studied. We studied how immune factors affect the function and epigenetics of beta cells during disease progression and identified possible triggers of these changes. METHODS: We studied FACS sorted beta cells and infiltrating lymphocytes from NOD mouse and human islets. Gene expression was measured by quantitative real-time RT-PCR (qRT-PCR) and methylation of the insulin genes was investigated by high-throughput and Sanger sequencing. To understand the role of DNA methyltransferases, Dnmt3a was knocked down with small interfering RNA (siRNA). The effects of cytokines on methylation and expression of the insulin gene were studied in humans and mice. RESULTS: During disease progression in NOD mice, there was an inverse relationship between the proportion of infiltrating lymphocytes and the beta cell mass. In beta cells, methylation marks in the Ins1 and Ins2 genes changed over time. Insulin gene expression appears to be most closely regulated by the methylation of Ins1 exon 2 and Ins2 exon 1. Cytokine transcription increased with age in NOD mice, and these cytokines could induce methylation marks in the insulin DNA by inducing methyltransferases. Similar changes were induced by cytokines in human beta cells in vitro. CONCLUSIONS/INTERPRETATION: Epigenetic modification of DNA by methylation in response to immunological stressors may be a mechanism that affects insulin gene expression during the progression of type 1 diabetes.

D-chiro-inositol glycan reduces food intake by regulating hypothalamic neuropeptide expression via AKT-FoxO1 pathway.

The regulation of food intake is important for body energy homeostasis. Hypothalamic insulin signaling decreases food intake by upregulating the expression of anorexigenic neuropeptides and downregulating the expression of orexigenic neuropeptides. INS-2, a Mn(2+) chelate of 4-O-(2-amino-2-deoxy-ß-D-galactopyranosyl)-3-O-methyl-D-chiro-inositol, acts as an insulin mimetic and sensitizer. We found that intracerebroventricular injection of INS-2 decreased body weight and food intake in mice. In hypothalamic neuronal cell lines, INS-2 downregulated the expression of neuropeptide Y (NPY), an orexigenic neuropeptide, but upregulated the expression of proopiomelanocortin (POMC), an anorexigenic neuropeptide, via modulation of the AKT-forkhead box-containing protein-O1 (FoxO1) pathway. Pretreatment of these cells with INS-2 enhanced the action of insulin on downstream signaling, leading to a further decrease in NPY expression and increase in POMC expression. These data indicate that INS-2 reduces food intake by regulating the expression of the hypothalamic neuropeptide genes through the AKT-FoxO1 pathway downstream of insulin.

Angiography reveals novel features of the retinal vasculature in healthy and diabetic mice.

The mouse retina is a commonly used animal model for the study of pathogenesis and treatment of blinding retinal vascular diseases such as diabetic retinopathy. In this study, we aimed to characterize normal and pathological variations in vascular anatomy in the mouse retina using fluorescein angiography visualized with scanning laser ophthalmoscopy and optical coherence tomography (SLO-OCT). We examined eyes from C57BL/6J wild type mice as well as the Ins2(Akita) and Akimba mouse models of diabetic retinopathy using the Heidelberg Retinal Angiography (HRA) and OCT system. Angiography was performed on three focal planes to examine distinct vascular layers. For comparison with angiographic data, ex vivo analyses, including Indian ink angiography, histology and 3D confocal scanning laser microscopy were performed in parallel. All layers of the mouse retinal vasculature could be readily visualized during fluorescein angiography by SLO-OCT. Blood vessel density was increased in the deep vascular plexus (DVP) compared with the superficial vascular plexus (SVP). Arteriolar and venular typologies were established and structural differences were observed between venular types. Unexpectedly, the hyaloid artery was found to persist in 15% of C57BL/6 mice, forming anastomoses with peripheral retinal capillaries. Fluorescein leakage was easily detected in Akimba retinae by angiography, but was not observed in Ins2(Akita) mice. Blood vessel density was increased in the DVP of 6 month old Ins2(Akita) mice, while the SVP displayed reduced branching in precapillary arterioles. In summary, we present the first comprehensive characterization of the mouse retinal vasculature by SLO-OCT fluorescein angiography. Using this clinical imaging technique, we report previously unrecognized variations in C57BL/6J vascular anatomy and novel features of vascular retinopathy in the Ins2(Akita) mouse model of diabetes.

Absence of clinical correlates of diabetic retinopathy in the Ins2Akita retina.

BACKGROUND: The Ins2(Akita) mouse has been reported to display retinal pathology degeneration associated with advanced diabetic retinopathy. In the present study, we monitored retinal changes in these mice to establish if this model displays clinical features associated with advanced diabetic retinopathy in human patients. METHODS: Ins2(Akita) mice (n = 55) on a C57Bl/6J background were monitored clinically from 9 to 25 weeks of age using a combination of scanning laser ophthalmoscopy, fluorescein angiography and optical coherence tomography. After clinical imaging, eyes were processed for immunostaining to examine microglial, astroglial and Muller glial responses to hyperglycaemia. To complement our optical coherence tomography imaging, retinal morphology and thicknesses were examined in high-quality semi-thin sections. RESULTS: No retinal thinning or disruption of retinal architecture was observed by optical coherence tomography or resin histology in Ins2(Akita) mice up to 6 months of age. In addition, no vascular changes were detected by fluorescein angiography or by scanning laser ophthalmoscopy. With the exception of microglial activation, reduced glial fibrillary acid protein expression in astrocytes and an increase in glial fibrillary acid protein expression by Muller cells, no other changes were observed in the Ins2(Akita) retina. CONCLUSIONS: Our results indicate that the classical clinical correlates of human diabetic retinopathy are absent in Ins2(Akita) mice up to 6 months of age suggesting that either the histopathological processes underlying the development of diabetic retinopathy in this model require longer than 5 months of hyperglycaemia to result in disruption of retinal architecture or that advanced diabetic retinopathy is not a feature of the Ins2(Akita) diabetic mouse.

Endogenous insulin directly protects pancreatic acinar cells in pancreatitis.

Diabetes is known to predispose patients to the development of the life-threatening disorder pancreatitis and to cause a more severe course of pancreatitis. However, the mechanistic link between diabetes and pancreatitis is not clear. Aberrant cytosolic Ca2+ signals within the main parenchymal cell of the pancreas, the acinar cells, are central to the initiation of pancreatitis and can modulate its severity. The acinar cell Ca2+ signals are tightly regulated by a Ca2+ toolbox, which includes the plasma membrane Ca2+-ATPase (PMCA). A new paper by Bruce et al. shows that active extrusion of Ca2+through the PMCA protects acinar cells against the damage of pancreatitis in the setting of diabetes. The novelty of the finding here is that insulin receptors on the acinar cell transduce a glycolytic supply of ATP to fuel the PMCA and, thereby, link diabetes to pancreatitis through Ca2+signaling.

A combined in vivo and in silico model shows specific predictors of individual trans-generational diabetic programming.

Diabetic pregnancies are cleary associated with maternal type 2 diabetes and metabolic syndrome as well as atherosclerotic diseases in the offspring. The global prevalence of hyperglycemia in pregnancy was estimated as 15.8% of live births to women in 2019, with an upward trend. Numerous parental risk factors as well as trans-generational mechanisms targeting the utero-placental system, leading to diabetes, dysmetabolic and atherosclerotic conditions in the next generation, seem to be involved within this pathophysiological context. To focus on the predictable impact of trans-generational diabetic programming, we studied age- and gender-matched offspring of diabetic and nondiabetic mothers. The offspring generation consists of three groups: C57BL/6-J-Ins2Akita (positive control group), wild-type C57BL/6-J-Ins2Akita (experimental group), and C57BL/6-J mice (negative control group). We undertook intraperitoneal glucose tolerance tests at 3 and 11 weeks of age. Moreover, this in vivo model was complemented by a corresponding in silico model. Although at 3 weeks of age, no significant effects could be observed, we could demonstrate at 11 weeks of age characteristic and significant differences in relation to maternal diabetic imprinting based on the in silico model-based predictors. These predictors allow the generation of a concise classification tree assigning maternal diabetic imprinting correctly in 91% of study cases. Our data show that hyperglycemic in utero milieu contributes to trans-generational diabetic programming leading to impaired glucose tolerance in the offspring of diabetic mothers early on. These observations can be clearly and early distinguished from genetically determined diabetes, for example, type 1 diabetes, in which basal glucose values are significantly raised.

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

IMPACT STATEMENT: 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 ß 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 ß-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 ß-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.