Ins2 Akita Mice Research Articles

TNFα Is Required for Late BRB Breakdown in Diabetic Retinopathy, and Its Inhibition Prevents Leukostasis and Protects Vessels and Neurons from Apoptosis

Blood-retinal barrier [BRB] breakdown, characteristic of diabetic retinopathy (DR), is believed to depend on inflammation and apoptosis. Retinal inflammation is almost completely suppressed in the absence of TNFα, which is also associated with apoptosis. This study was conducted to determine the role of TNFα in these diabetic complications. Diabetes was induced with streptozotocin in Tnfa knockout (KO) mice, to provide a chemical model of diabetes, and Tnfa (KO) mice were crossed with Ins2(Akita) mice to generate a genetic model, with both models being devoid of TNFα. The BRB was assessed at 1, 1.5, 3, and 6 months. Leukostasis was assessed using FITC-conjugated ConA to label leukocytes. Apoptosis was assessed with TUNEL and activated caspase-3 staining. PECAM1 identified endothelial cells, and SMA identified pericytes. At 1 month of diabetes, the absence of TNFα had no effect on DR-associated BRB breakdown, even though it prevented retinal leukostasis, demonstrating that neither TNFα nor inflammation is essential for early BRB breakdown in DR in either model of diabetes. At 3 months of diabetes, BRB breakdown was significantly suppressed and at 6 months, it was completely prevented in the absence of TNFα in both models, showing that TNFα is essential for progressive BRB breakdown. DR-mediated apoptosis in the retina, which appears to involve endothelial cells, pericytes, and neurons, was inhibited in the absence of TNFα in both models. Although neither TNFα nor inflammation is necessary for early BRB breakdown in DR, TNFα is critical for later complications and would be a good therapeutic target for the prevention of the progressive BRB breakdown, retinal leukostasis, and apoptosis associated with DR.

Human Immune System Development and Rejection of Human Islet Allografts in Spontaneously Diabetic NOD-Rag1null IL2rγnull Ins2Akita Mice

OBJECTIVETo create an immunodeficient mouse model that spontaneously develops hyperglycemia to serve as a diabetic host for human islets and stem cell–derived β-cells in the absence or presence of a functional human immune system.RESEARCH DESIGN AND METHODSWe backcrossed the Ins2Akita mutation onto the NOD-Rag1null IL2rγnull strain and determined 1) the spontaneous development of hyperglycemia, 2) the ability of human islets, mouse islets, and dissociated mouse islet cells to restore euglycemia, 3) the generation of a human immune system following engraftment of human hematopoietic stem cells, and 4) the ability of the humanized mice to reject human islet allografts.RESULTSWe confirmed the defects in innate and adaptive immunity and the spontaneous development of hyperglycemia conferred by the IL2rγnull, Rag1null, and Ins2Akita genes in NOD-Rag1null IL2rγnull Ins2Akita (NRG-Akita) mice. Mouse and human islets restored NRG-Akita mice to normoglycemia. Insulin-positive cells in dissociated mouse islets, required to restore euglycemia in chemically diabetic NOD-scid IL2rγnull and spontaneously diabetic NRG-Akita mice, were quantified following transplantation via the intrapancreatic and subrenal routes. Engraftment of human hematopoietic stem cells in newborn NRG-Akita and NRG mice resulted in equivalent human immune system development in a normoglycemic or chronically hyperglycemic environment, with >50% of engrafted NRG-Akita mice capable of rejecting human islet allografts.CONCLUSIONSNRG-Akita mice provide a model system for validation of the function of human islets and human adult stem cell, embryonic stem cell, or induced pluripotent stem cell–derived β-cells in the absence or presence of an alloreactive human immune system.

Hyperglycemia Impairs Proteasome Function by Methylglyoxal

OBJECTIVEThe ubiquitin-proteasome system is the main degradation machinery for intracellularly altered proteins. Hyperglycemia has been shown to increase intracellular levels of the reactive dicarbonyl methylglyoxal (MGO) in cells damaged by diabetes, resulting in modification of proteins and alterations of their function. In this study, the influence of MGO-derived advanced glycation end product (AGE) formation on the activity of the proteasome was investigated in vitro and in vivo.RESEARCH DESIGN AND METHODSMGO-derived AGE modification of proteasome subunits was analyzed by mass spectrometry, immunoprecipitation, and Western blots. Proteasome activity was analyzed using proteasome-specific fluorogenic substrates. Experimental models included bovine retinal endothelial cells, diabetic Ins2Akita mice, glyoxalase 1 (GLO1) knockdown mice, and streptozotocin (STZ)-injected diabetic mice.RESULTSIn vitro incubation with MGO caused adduct formation on several 20S proteasomal subunit proteins. In cultured endothelial cells, the expression level of the catalytic 20S proteasome subunit was not altered but proteasomal chymotrypsin-like activity was significantly reduced. In contrast, levels of regulatory 19S proteasomal proteins were decreased. In diabetic Ins2Akita, STZ diabetic, and nondiabetic and diabetic G101 knockdown mice, chymotrypsin-like activity was also reduced and MGO modification of the 20S-β2 subunit was increased.CONCLUSIONSHyperglycemia-induced formation of MGO covalently modifies the 20S proteasome, decreasing its activity in the diabetic kidney and reducing the polyubiquitin receptor 19S-S5a. The results indicate a new link between hyperglycemia and impairment of cell functions.

HIGH‐CHOLESTEROL DIET AUGMENTS ENDOTHELIAL DYSFUNCTION VIA ELEVATED OXIDATIVE STRESS AND REDUCED TETRAHYDROBIOPTERIN IN INS2AKITA MICE, AN AUTOSOMAL DOMINANT MUTANT TYPE 1 DIABETIC MODEL

1. Oxidative stress contributes to endothelial dysfunction and atherogenesis in diabetes. The present study tested the hypothesis that a high-cholesterol diet accelerates endothelial dysfunction in Ins2(Akita) mice, a Type 1 diabetic model with a spontaneous autosomal preproinsulin gene (Ins2 gene) mutation, through further increase of superoxide production. 2. The Ins2(Akita) diabetic mice were fed a high-cholesterol diet (1.25% cholesterol) for 4 months. Some Ins2(Akita) mice were also treated for 4 months with the selective NADPH oxidase inhibitor apocynin (4 mg/kg per day in drinking water). Oxidative stress markers, tetrahydrobiopterin (BH4) levels, GTP cyclohydrolase I activity and endothelial function were determined in serum or arteries afterwards. 3. Serum lipid peroxidation and arterial superoxide levels were increased, whereas arterial BH(4) levels and GTP cyclohydrolase I activity were decreased, in Ins2(Akita) mice on a high-cholesterol diet, resulting in impaired endothelium-dependent nitric oxide-mediated relaxation in response to acetylcholine. 4. In vivo treatment with apocynin not only blunted serum lipid peroxidation and arterial superoxide levels, but also increased BH4 levels and GTP cyclohydrolase I activity, resulting in improved endothelium-dependent relaxation. 5. These results suggest that NADPH oxidase may play a potential role in oxidative stress-induced arterial BH4 and GTP cyclohydrolase I deficiency, resulting in endothelial dysfunction in Ins2(Akita) Type 1 diabetic mice fed a high-cholesterol diet.

Neuritic dystrophy and neuronopathy in Akita ( Ins2Akita) diabetic mouse sympathetic ganglia

Diabetic autonomic neuropathy is a debilitating, poorly studied complication of diabetes. Our previous studies of non-obese diabetic (NOD) and related mouse models identified rapidly developing, dramatic pathology in prevertebral sympathetic ganglia; however, once diabetic, the mice did not survive for extended periods needed to examine the ability of therapeutic agents to correct established neuropathy. In the current manuscript we show that the Akita ( Ins2 Akita ) mouse is a robust model of diabetic sympathetic autonomic neuropathy with unambiguous, spontaneous, rapidly-developing neuropathology which corresponds closely to the characteristic pathology of other rodent models and man. Akita mice diabetic for 2, 4 or 8 months of diabetes progressively developed markedly swollen axons and dendrites (“neuritic dystrophy”) in the prevertebral superior mesenteric (SMG) and celiac ganglia (CG). Comparable changes failed to develop in the superior cervical ganglia (SCG) of the Akita mouse or in any ganglia of non-diabetic mice. Morphometric studies demonstrate an overall increase in presynaptic axon terminal cross sectional area, including those without any ultrastructural features of dystrophy. Neurons in Akita mouse prevertebral sympathetic ganglia show an unusual perikaryal alteration characterized by the accumulation of membranous aggregates and minute mitochondria and loss of rough endoplasmic reticulum. These changes result in the loss of a third of neurons in the CG over the course of 8 months of diabetes. The extended survival of diabetic mice and robust pathologic findings provide a clinically relevant paradigm that will facilitate the analysis of novel therapeutic agents on the reversal of autonomic neuropathy.

INCREASED SUPEROXIDE CONTRIBUTES TO ENHANCEMENT OF VASCULAR CONTRACTION IN INS2AKITA DIABETIC MICE, AN AUTOSOMAL DOMINANT MUTANT MODEL

Superoxide has been reported to be involved in vascular dysfunction in diabetes. The Ins2(Akita) mouse is an autosomal dominant mutant diabetic model that can serve as an excellent substitute for the Type 1 diabetic mouse model induced by chemical diabetogens. The purpose of the present study was to investigate the role of superoxide on vascular dysfunction using this new diabetic model. Compared with age-matched normal C57BL/6 mice, in Ins2(Akita) diabetic mice arterial superoxide, lipid peroxidation production (1.2 +/- 0.1 vs 17.4 +/- 1.9 mmol/mg tissue, respectively; P < 0.01) and plasma lipid peroxidation production (0.08 +/- 0.02 vs 0.40 +/- 0.03 mmol/L, respectively; P < 0.01) were increased. Meanwhile, expression of vascular adhesion molecule-1, E-selectin and monocyte chemoattractant protein-1 in the aorta and/or plasma was elevated. The contraction of carotid arteries to U46619 in Ins2(Akita) diabetic mice was significantly enhanced compared with control mice (P < 0.05). Tempol (a scavenger of superoxide), apocynin (an inhibitor of NADPH oxidase) and allopurinol (an inhibitor of xanthine oxidase) all not only decreased superoxide in carotid arteries, but also suppressed arterial contractions to U46619 in Ins2(Akita) diabetic mice. Indomethacin, an inhibitor of cyclo-oxygenase, and chelerythrine, an inhibitor of protein kinase C, also suppressed the enhanced vascular contraction. These results suggest that increased arterial superoxide generated from diverse sources may potentiate the contractions of carotid arteries in Ins2(Akita) diabetic mice.

A new immunodeficient hyperglycaemic mouse model based on the Ins2Akita mutation for analyses of human islet and beta stem and progenitor cell function.

To develop and validate a new immunodeficient mouse strain that spontaneously develops a non-autoimmune hyperglycaemia to serve as a diabetic host for human islets and human beta stem and progenitor cells without the need for induction of hyperglycaemia by toxic chemicals with their associated side effects. We generated and characterised a new strain of immunodeficient spontaneously hyperglycaemic mice, the NOD-Rag1null Prf1null Ins2Akita strain and compared this strain with the NOD-scid Il2rgammanull (also known as Il2rg) immunodeficient strain rendered hyperglycaemic by administration of a single dose of streptozotocin. Hyperglycaemic mice were transplanted with human islets ranging from 1,000 to 4,000 islet equivalents (IEQ) and were monitored for normalisation of blood glucose levels. NOD-Rag1null Prf1null Ins2Akita mice developed spontaneous hyperglycaemia, similar to Ins2Akita-harbouring strains of immunocompetent mice. Histological examination of islets in the host pancreas validated the spontaneous loss of beta cell mass in the absence of mononuclear cell infiltration. Human islets transplanted into spontaneously diabetic NOD-Rag1null Prf1null Ins2Akita and chemically diabetic NOD-scid Il2rgammanull mice resulted in a return to euglycaemia that occurred with transplantation of similar beta cell masses. The NOD-Rag1null Prf1null Ins2Akita mouse is the first immunodeficient, spontaneously hyperglycaemic mouse strain described that is based on the Ins2Akita mutation. This strain is suitable as hosts for human islet and human beta stem and progenitor cell transplantation in the absence of the need for pharmacological induction of diabetes. This strain of mice also has low levels of innate immunity and can be engrafted with a human immune system for the study of human islet allograft rejection.

Maternal diabetes causes coordinated down-regulation of genes involved with lipid metabolism in the murine fetal heart

Maternal diabetes is associated with increased transport of lipids to the fetus and increased risk of hypertrophic cardiomyopathy in the fetus. During fetal life, the heart normally has limited capacity to use lipids as fuel; and, at least in adults, cardiac lipid accumulation may lead to cardiomyopathy. Postnatally, lipid supply is increased when the offspring begins to suckle. We examined offspring from hypoinsulinemic Ins2 Akita mice to assess whether maternal diabetes results in fetal myocardial hypertrophy and triglyceride accumulation and compared these with fetal hearts collected postnatally. On embryonic days 16 to 19, the fetal heart weight and triglyceride content were similar in offspring from Ins2 Akita and nondiabetic wild-type mothers. The heart expression of lipid-metabolizing genes (peroxisomal proliferator-activated receptor α, lipoprotein lipase, fatty acid translocase, and fatty acid transport protein 1) was reduced in offspring from Ins2 Akita mothers with high blood glucose levels and were closely intercorrelated, suggesting coordinated down-regulation. In contrast, on day 1 postnatally where the lipid availability to the heart is markedly increased, heart triglycerides and expression of several lipid-metabolizing genes (including lipoprotein lipase and fatty acid transport protein 1) were increased in offspring from wild-type mice. The results suggest that maternal type 1 diabetes mellitus in Ins2 Akita mice does not cause cardiac hypertrophy or triglycerides accumulation in the fetal heart, possibly because of a coordinated down-regulation of genes controlling fatty acid uptake.

The Role of Osteopontin in the Development of Albuminuria

Several gene array studies have suggested that osteopontin (Opn) expression strongly correlates with albuminuria and glomerular disease. Urinary Opn concentration and kidney Opn immunoreactivity were found to be increased in patients with steroid-sensitive nephrotic syndrome. In addition, renal Opn mRNA was increased in the Ins2(Akita) mouse model of type 1 diabetic nephropathy, in the LPS-induced albuminuria model, and in glomeruli of puromycin aminonucleotide-induced nephrotic rats. Opn knockout mice did not develop albuminuria in response to LPS injection, and Opn knockout mice were protected from diabetes-induced albuminuria and mesangial expansion. In the glomerulus, Opn immunostaining was increased specifically in podocytes. Treatment of podocytes with recombinant Opn activated the NF-kappaB pathway, increased expression of urokinase plasminogen activator and matrix metalloproteinases 2 and 9, and increased podocyte motility. Taken together, these results indicate that Opn plays an important role in the development of albuminuria, possibly by modulating podocyte signaling and motility.

Ins2Akita mice exhibit hyperphagia and anxiety behavior via the melanocortin system

Elevated anxiety symptoms have been reported to be present in many patients with diabetes mellitus. The underlying mechanisms by which diabetes mellitus influences behavior remain to be determined. We assessed feeding and anxiety behaviors in spontaneously diabetic Ins2Akita mice. We measured blood glucose, body weight, and food and water intakes in C57BL/6 heterozygote Ins2Akita mice. The behavioral properties of Ins2Akita mice were assessed in an open-field test and an elevated plus-maze. The gene expression of hypothalamic neuropeptides was examined in non-food-deprived Ins2Akita mice. Body weights of the Ins2Akita mice were less than those of the age-matched C57BL/6 mice, as controls. Food and water intakes were increased in the Ins2Akita mice. In the open-field test, the Ins2Akita mice had decreased locomotor activity and increased immobilization time. The Ins2Akita mice exhibited anxiety behavior in the elevated plus-maze. RT-PCR analysis showed decreased proopiomelanocortin (POMC) mRNA expression and increased agouti-related protein (AGRP) mRNA expression in Ins2Akita mice. There were no significant differences in hypothalamic ghrelin mRNA expression. These observations indicate that Ins2Akita mice, which are characterized by hypoinsulinemia and hyperglycemia, exhibited hyperphagia and anxiety behavior; the mechanism of action involved the activation of hypothalamic AGRP and the inactivation of hypothalamic POMC. In addition, Ins2Akita mice are a useful model for understanding the mechanisms involved in the psychological complications of diabetes mellitus. Further, melanocortin systems may be therapeutic targets not only for diabetes but also for its associated complications.

Glucose-Induced Reactive Oxygen Species Cause Apoptosis of Podocytes and Podocyte Depletion at the Onset of Diabetic Nephropathy

Diabetic nephropathy is the most common cause of end-stage renal disease in the U.S. Recent studies demonstrate that loss of podocytes is an early feature of diabetic nephropathy that predicts its progressive course. Cause and consequences of podocyte loss during early diabetic nephropathy remain poorly understood. Here, we demonstrate that podocyte apoptosis increased sharply with onset of hyperglycemia in Ins2(Akita) (Akita) mice with type 1 diabetes and Lepr(db/db) (db/db) mice with obesity and type 2 diabetes. Podocyte apoptosis coincided with the onset of urinary albumin excretion (UAE) and preceded significant losses of podocytes in Akita (37% reduction) and db/db (27% reduction) mice. Increased extracellular glucose (30 mmol/l) rapidly stimulated generation of intracellular reactive oxygen species (ROS) through NADPH oxidase and mitochondrial pathways and led to activation of proapoptotic p38 mitogen-activated protein kinase and caspase 3 and to apoptosis of conditionally immortalized podocytes in vitro. Chronic inhibition of NADPH oxidase prevented podocyte apoptosis and ameliorated podocyte depletion, UAE, and mesangial matrix expansion in db/db mice. In conclusion, our results demonstrate for the first time that glucose-induced ROS production initiates podocyte apoptosis and podocyte depletion in vitro and in vivo and suggest that podocyte apoptosis/depletion represents a novel early pathomechanism(s) leading to diabetic nephropathy in murine type 1 and type 2 diabetic models.

The Ins2AkitaMouse as a Model of Early Retinal Complications in Diabetes

This study tested the Ins2(Akita) mouse as an animal model of retinal complications in diabetes. The Ins2(Akita) mutation results in a single amino acid substitution in the insulin 2 gene that causes misfolding of the insulin protein. The mutation arose and is maintained on the C57BL/6J background. Male mice heterozygous for this mutation have progressive loss of beta-cell function, decreased pancreatic beta-cell density, and significant hyperglycemia, as early as 4 weeks of age. Heterozygous Ins2(Akita) mice were bred to C57BL/6J mice, and male offspring were monitored for hyperglycemia, beginning at 4.5 weeks of age. After 4 to 36 weeks of hyperglycemia, the retinas were analyzed for vascular permeability, vascular lesions, leukostasis, morphologic changes of micro- and macroglia, apoptosis, retinal degeneration, and insulin receptor kinase activity. The mean blood glucose of Ins2(Akita) mice was significantly elevated, whereas the body weight at death was reduced compared with that of control animals. Compared with sibling control mice, the Ins2(Akita) mice had increased retinal vascular permeability after 12 weeks of hyperglycemia (P < 0.005), a modest increase in acellular capillaries after 36 weeks of hyperglycemia (P < 0.0008), and alterations in the morphology of astrocytes and microglia, but no changes in expression of Muller cell glial fibrillary acidic protein. Increased apoptosis was identified by immunoreactivity for active caspase-3 after 4 weeks of hyperglycemia (P < 0.01). After 22 weeks of hyperglycemia, there was a 16.7% central and 27% peripheral reduction in the thickness of the inner plexiform layer, a 15.6% peripheral reduction in the thickness of the inner nuclear layer (P < 0.001), and a 23.4% reduction in the number of cell bodies in the retinal ganglion cell layer (P < 0.005). In vitro insulin receptor kinase activity was reduced (P < 0.05) after 12 weeks of hyperglycemia. The retinas of heterozygous male Ins2(Akita) mice exhibit vascular, neural, and glial abnormalities generally consistent with clinical observations and other animal models of diabetes. In light of the relatively early, spontaneous onset of the disease and the popularity of the C57BL/6J inbred strain as a background for the generation and study of other genetic alterations, combining the Ins2(Akita) mutation with other engineered mutations will be of great use for studying the molecular basis of retinal complications of diabetes.