Experimental Diabetes In Mice Research Articles

Дефіцит вітаміну D3 та його роль у розвитку метаболічних порушень

Подано дані про вплив вітаміну D3 на кальцій-фосфорний і вуглеводний обміни, а також на функціональну активність β-клітин підшлункової залози на тлі D-гіповітамінозу та при експериментальному діабеті в мишей. Описано деякі механізми порушення обміну вітаміну D3 і результати його клінічного застосування в пацієнтів із цукровим діабетом 1-го і 2-го типу.

Endothelial mineralocorticoid receptor ablation confers protection towards endothelial dysfunction in experimental diabetes in mice.

With diabetes comes a significant risk of macrovascular and microvascular complications. Circulating aldosterone levels increase in patients with diabetes. Aldosterone can directly affect vascular function via activation of the mineralocorticoid receptor (MR). We hypothesized that aldosterone via endothelial MR impairs endothelial function in a murine model of experimental diabetes. Endothelial cell-specific mineralocorticoid receptor knockout MRflox/flox ; Tie2-Cre mice (ECMR-KO) and wild-type FVB littermates were subjected to an experimental type-1 diabetic model by low dose streptozotocin injections (55mg/kg/day) for five consecutive days. After 10weeks of diabetes, second-order mesenteric resistance arteries were perfused ex vivo to evaluate vessel contractility and endothelial function. The effect of ex vivo incubation with aldosterone with and without the antagonist, spironolactone was determined. Diabetic ECMR-KO and wild-type mice had similar, elevated, plasma aldosterone concentration while only diabetic wild-type mice displayed elevated urine albumin excretion and cardiac and kidney hypertrophy at 10weeks. There were no differences in contraction (Emax and EC50 ) to thromboxane receptor agonist (U46619) and elevated K+ between groups. Wild-type diabetic mice showed impaired acetylcholine (ACh)-dependent relaxation, while diabetic ECMR-KO mice had intact ACh-mediated relaxation. Aldosterone incubation ex vivo impaired ACh mediated relaxation and rendered responses similar to diabetic WT arteries. Direct, ex vivo aldosterone effects were absent in ECMR-KO animals. Ex vivo inhibitory effects of aldosterone on endothelial relaxation in arteries from WT were abolished by spironolactone. These findings show that endothelial cell mineralocorticoid receptor activation accounts for diabetes-induced systemic endothelial dysfunction in experimental diabetes and may explain the cardiovascular protection by MR antagonists in diabetes.

Construction of diabetic mice model carrying external wound for the evaluation of topical applications on the impaired wound healing

Chronic leg ulcer is one of the diabetic complications causing continuous pain and higher mortality risk. The therapeutic treatment of chronic diabetic wounds can be studied through animal models which simulate the human pathology. Experimental diabetes in mice induced by streptozotocin (STZ) is an easy method with low cost. There have been several studies using STZ for diabetic mice model of cutaneous excisional wound. However, those procedures are different in many factors such as dose of STZ, mouse weight, blood glucose level and the method for assessing health status in mice. We have constructed the criteria to evaluate the health status of the experimental mice including skin color, fur condition, ability to move and body condition. These criteria of health status wereused together with the blood glucose level to evaluate the male mice’s sensitivity to a single high dose (100-150 mg/kg) of STZ. Approximately 40 % of 20–28 g weighted mice, which were injected by 125 mg/kg STZ, developed diabetes in a good health status in the first two weeks after injection. Mice with blood glucose level higher than 200 mg/dL had damaged islet and stably high blood glucose as well as stable health status after two weeks carrying wounds. The wound healing process of the diabetic wounded mice occured relatively slower than that of the control group (non diabetic). The model of diabetic wounded mice has been used to evaluate of the wound healing effect of the rhPDGF-BB protein which has been used in the treatment of chronic wounds caused by human diabetes.

Deficiency of S100B confers resistance to experimental diabetes in mice

The calcium binding protein S100B has been implicated in diabetic neuronal and vascular complications but has not been examined in the development of diabetes. S100B knock out (S100B KO) and wild-type (WT) mice were injected with 40 mg/kg body weight streptozotocin (STZ) for 5 days. Blood and pancreatic tissue samples were obtained to examine islet structure and function, the profile of glucose and insulin and expression of glucose transporter 2 (Glut2), S100B and its receptor, the receptor for advanced glycation end products (RAGE). Primary islet β-cells cultures from WT mice were used to test the apoptotic potential of S100B. S100B KO mice were resistant to STZ induced-diabetes with lower urine volume, food and water intake compared to WT mice. S100B increased in the WT islet following diabetes but did not co-localize with beta or peri-islet Schwann cells but with CD3 + T lymphocytes. S100B KO mice exhibited enhanced glucose tolerance, insulin sensitivity, prevented β-cell destruction and functional impairment in response to STZ treatment. S100B deficiency was associated with decreased Glut2 and RAGE. In primary β-cell cultures from WT mice, S100B induced reactive oxygen species (ROS) and RAGE-dependent apoptosis. In the STZ diabetic animal model, abrogation of S100B enhances insulin sensitivity and reduces pancreatic islet, and β-cell destruction. S100B may be a promising target for pharmacological interventions aimed at repressing diabetes.

Monomeric eNAMPT in the development of experimental diabetes in mice: a potential target for type 2 diabetes treatment.

Aims/hypothesisSerum extracellular nicotinamide phosphoribosyltransferase (eNAMPT) concentrations are elevated in type 2 diabetes. However, the relationship between abnormally elevated serum eNAMPT and type 2 diabetes pathophysiology is unclear. eNAMPT circulates in functionally and structurally distinct monomeric and dimeric forms. Dimeric eNAMPT promotes NAD biosynthesis. The role of eNAMPT-monomer is unclear but it may have NAD-independent proinflammatory effects. However, studies of eNAMPT in type 2 diabetes have not distinguished between monomeric and dimeric forms. Since type 2 diabetes is characterised by chronic inflammation, we hypothesised a selective NAD-independent role for eNAMPT-monomer in type 2 diabetes.MethodsTwo mouse models were used to examine the role of eNAMPT-monomer in type 2 diabetes; (1) a mouse model of diabetes fed a high-fat diet (HFD) for 10 weeks received i.p. injections with an anti-monomeric-eNAMPT antibody; and (2) lean non-diabetic mice received i.p. injections with recombinant monomeric eNAMPT daily for 14 days.ResultsSerum monomeric eNAMPT levels were elevated in HFD-fed mouse models of diabetes, whilst eNAMPT-dimer levels were unchanged. eNAMPT-monomer neutralisation in HFD-fed mice resulted in lower blood glucose levels, amelioration of impaired glucose tolerance (IGT) and whole-body insulin resistance, improved pancreatic islet function, and reduced inflammation. These effects were maintained for at least 3 weeks post-treatment. eNAMPT-monomer administration induced a diabetic phenotype in mice, characterised by elevated blood glucose, IGT, impaired pancreatic insulin secretion and the presence of systemic and tissue inflammation, without changes in NAD levels.Conclusions/interpretationWe demonstrate that elevation of monomeric-eNAMPT plays an important role in the pathogenesis of diet-induced diabetes via proinflammatory mechanisms. These data provide proof-of-concept evidence that the eNAMPT-monomer represents a potential therapeutic target for type 2 diabetes.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-016-4076-3) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Islet-Like Cell Aggregates Generated from Human Adipose Tissue Derived Stem Cells Ameliorate Experimental Diabetes in Mice

BackgroundType 1 Diabetes Mellitus is caused by auto immune destruction of insulin producing beta cells in the pancreas. Currently available treatments include transplantation of isolated islets from donor pancreas to the patient. However, this method is limited by inadequate means of immuno-suppression to prevent islet rejection and importantly, limited supply of islets for transplantation. Autologous adult stem cells are now considered for cell replacement therapy in diabetes as it has the potential to generate neo-islets which are genetically part of the treated individual. Adopting methods of islet encapsulation in immuno-isolatory devices would eliminate the need for immuno-suppressants.Methodology/Principal FindingsIn the present study we explore the potential of human adipose tissue derived adult stem cells (h-ASCs) to differentiate into functional islet like cell aggregates (ICAs). Our stage specific differentiation protocol permit the conversion of mesodermic h-ASCs to definitive endoderm (Hnf3β, TCF2 and Sox17) and to PDX1, Ngn3, NeuroD, Pax4 positive pancreatic endoderm which further matures in vitro to secrete insulin. These ICAs are shown to produce human C-peptide in a glucose dependent manner exhibiting in-vitro functionality. Transplantation of mature ICAs, packed in immuno-isolatory biocompatible capsules to STZ induced diabetic mice restored near normoglycemia within 3–4 weeks. The detection of human C-peptide, 1155±165 pM in blood serum of experimental mice demonstrate the efficacy of our differentiation approach.Conclusionsh-ASC is an ideal population of personal stem cells for cell replacement therapy, given that they are abundant, easily available and autologous in origin. Our findings present evidence that h-ASCs could be induced to differentiate into physiologically competent functional islet like cell aggregates, which may provide as a source of alternative islets for cell replacement therapy in type 1 diabetes.

Reversal of experimental diabetes in mice by transplantation of neo-islets generated from human amnion-derived mesenchymal stromal cells using immuno-isolatory macrocapsules

The ethical and biologic limitations with current sources of stem cells have resulted in a quest to look for alternative sources of multipotent stem cells of human origin. Amniotic membrane is of interest as a source of cells for regenerative medicine because of its ease of availability, plasticity and inexhaustible source that does not violate the sanctity of independent life. Although researchers have shown the stem cell-like potential of human amniotic epithelial cells, the mesenchymal part of amnion has remained less explored. We established a long-term culture of mesenchymal-like stem cells derived from full-term human amniotic membrane and their differentiation into functional pancreatic lineage. The amnion-derived mesenchymal-like stem cells expressed various mesenchymal markers and demonstrated multilineage differentiation capacity. We also observed that these cells could form islet-like clusters (ILC) on exposure to serum-free defined media containing specific growth factor and differentiating agents. Differentiated ILC showed expression of human insulin, glucagon and somatostatin by immunocytochemistry, while quantitative reverse transcription/real-time-polymerase chain reaction (qRT-PCR) data demonstrated the expression of insulin, glucagon, somatostatin, Ngn3 and Isl1. Moreover, encapsulation of the ILC in polyurethane-polyvinyl pyrrolidone macrocapsules and their subsequent transplantation in experimental diabetic mice resulted in restoration of normoglycemia, indicating their ability to respond to high glucose without immunorejection. Our results demonstrate that amnion-derived mesenchymal stromal cells can undergo islet neogenesis, indicating amnion as an alternative source of islets for cell replacement therapy in diabetes.

Beneficial effects of the ethanol extract from the dry matter of a culture broth of Inonotus obliquus in submerged culture on the antioxidant defence system and regeneration of pancreatic β-cells in experimental diabetes in mice

The antihyperglycaemic and antilipidperoxidative effects of the ethanol extract from the dry matter of a culture broth (DMCB) of Inonotus obliquus were investigated in alloxan-induced diabetic mice and the possible mechanism of action was also discussed. In alloxan-induced diabetic mice, treatment with the ethanol extract from DMCB of I. obliquus (30 and 60 mg kg−1 body weight (b.w.) for 21 days) showed a significant decrease in blood glucose level: the percentage reductions on the 7th day were 11.54 and 11.15%, respectively. However, feeding of this drug for three weeks produced reduction of 22.51 and 24.32%. Furthermore, the ethanol extract from the DMCB of I. obliquus treatment significantly decreased serum contents of free fatty acids, total cholesterol, triglycerides and low-density lipoprotein-cholesterol, whereas it effectively increased high-density lipoprotein-cholesterol, insulin levels and hepatic glycogen contents in livers of diabetic mice. Besides this, the ethanol extracts from the DMCB treatment significantly increased catalase, superoxide dismutase and glutathione peroxidase activities, except for decreasing the maleic dialdehyde level in diabetic mice. Histological morphology examination showed that the ethanol extract from the DMCB of I. obliquus restored the damage of pancreatic tissues in mice with diabetes mellitus. The results showed that the ethanol extract from the DMCB of I. obliquus possesses significant antihyperglycaemic, antilipidperoxidative and antioxidant effects in alloxan-induced diabetic mice.

Human Placenta-Derived Mesenchymal Stem Cells and Islet-Like Cell Clusters Generated From These Cells as a Novel Source for Stem Cell Therapy in Diabetes

Placental tissue holds great promise as a source of cells for regenerative medicine due to its plasticity, and easy availability. Human placenta-derived mesenchymal stem cells (hPDMSCs) have the potential to differentiate into insulin-producing cells. Upon transplantation, they can reverse experimental diabetes in mice. However, it is not known whether culture-expanded undifferentiated hPDMSCs are capable of restoring normoglycemia upon transplantation in streptozotocin (STZ)-induced diabetic mice. Hence we prepared long-term cultures of hPDMSCs from the chorionic villi of full-term human placenta. Flow cytometry analyses and immunocytochemistry study revealed bonafide mesenchymal nature of the isolated hPDMSCs. These cultures could differentiate into adipogenic, oesteogenic, chondrogenic, and neuronal lineages on exposure to lineage-specific cocktails. Furthermore, we showed that hPDMSCs can form islet-like cell clusters (ILCs) on stepwise exposure to serum-free defined media containing specific growth factors and differentiating agents. qRT-PCR showed the expression of insulin, glucagon, and somatostatin in undifferentiated hPDMSCs and in ILCs. Differentiated ILCs were found to express human insulin, glucagon, and somatostatin by immunocytochemistry. Additionally, ILCs also showed abundance of pancreatic transcription factors ngn3 and isl1. Both undifferentiated hPDMSCs and ILCs exihibited insulin secretion in response to glucose. Transplantation of hPDMSCs or ILCs derived from hPDMSCs in STZ-induced diabetic mice led to restoration of normoglycemia. Our results demonstrate, for the first time, reversal of hyperglycemia by undifferentiated hPDMSCs and ILCs derived from hPDMSCs. These results suggest human placenta-derived MSCs as an alternative source for cell replacement therapy in diabetes.

CR-6 protects glutathione peroxidase activity in experimental diabetes

Antioxidants can be useful as a supportive therapy in diabetes, and we try to elucidate some of the mechanisms by which these compounds are able to protect from diabetic complications. For this purpose we have assayed, in vitro and in vivo, the ability of CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2 H)-benzopyran), an antioxidant able to scavenge nitrogen reactive species, to protect glutathione peroxidase (GPx) activity. Glucose, in vitro, inhibited GPx activity in a concentration-dependent manner; CR-6 was able to protect GPx activity from glucose-induced inactivation. Alloxan-induced experimental diabetes in mice promoted oxidative stress in the retina and hippocampus, after 3 weeks of hyperglycemia. CR-6 administration prevented not only the alterations of oxidative stress markers (tissue GSH and malondialdehyde (MDA) concentration and GPx activity) but also the impairment of retinal function (as assessed by the modifications in electroretinogram b-wave amplitude). The findings herein confirm the role of nitrogen reactive species in diabetes; therefore, antioxidants effectively quenching these species, such as CR-6, should be considered for the adjuvant treatment of diabetes.

The Association of Acetyl-l-Carnitine and Nicotinamide Remits the Experimental Diabetes in Mice by Multiple Low-dose Streptozotocin

We studied the effect of acetyl-l-carnitine plus nicotinamide (AC + N) on murine diabetes mellitus induced by multiple low doses of streptozotocin. Male C57BL/6J inbred mice were injected intraperitoneally with citrate buffer or streptozotocin (40 mg/kg) for 5 consecutive days, followed by injections of saline solution or AC + N (50 + 25 mg/kg) from days 6 to 110. Four groups were studied: normal control mice (C), treated normal control mice (TC), diabetic mice (D), and treated diabetic mice (TD). TD group was divided into 2 at day 86; treatment was suspended in one group (TDs) and continued in the other until day 110. Weight, plasma glucose, plasma insulin, cellular immune aggression, glucose-stimulated insulin secretion from perifused pancreatic slices, and pancreas histology were studied in each experimental group. Diabetic mice treated with AC + N showed improvements in weight, plasma glucose, and plasma insulin levels without mortality, reaching control values at day 110. Cellular immune aggression and insulin release from pancreatic slices perfusions improved without reaching control values. Histology showed that insulin-immunostained area, the index of insulin immunostained beta cells and beta-cell size, was normalized at the end of the study. The treatment with AC + N induced remission of autoimmune type 1 diabetes in mice produced by multiple low doses of streptozotocin.

Neurite outgrowth in dorsal root ganglia induced by islet neogenesis-associated protein peptide involves protein kinase A activation

Islet neogenesis-associated protein (INGAP) peptide is a candidate therapeutic for diabetes and corrects sensory dysfunction in experimental diabetes in mice. In this study, we investigated the mechanism of action by which INGAP peptide promotes neurite outgrowth in sensory neurons of the dorsal root ganglia. Treatment of dorsal root ganglia primary dispersed cultures with INGAP peptide led to the displacement of fluorescently labeled forskolin from adenylate cyclase, the cyclic AMP-generating enzyme that has been implicated in neuritogenesis. The addition of forskolin or dibutyryl cyclic AMP enhanced the effects of INGAP peptide on neurite outgrowth in dorsal root ganglia explant cultures. Furthermore, pharmacological inhibition of adenylate cyclase with SQ22,536 or of protein kinase A with H89 or KT5720 significantly reduced the neurite-promoting effects of INGAP peptide. These results suggest that INGAP peptide-induced neurite outgrowth in the dorsal root ganglia partially involves cyclic AMP-dependent activation of protein kinase A.

Diabetes Mellitus in Cattle Infected With Bovine Viral Diarrhea Mucosal Disease Virus

Diabetes in cattle has various etiologies1. In humans, virus-induced insulin dependent diabetes mellitus (IDDM) has been reported. It has also been reported that cattle infected with foot-and-mouth disease contracted diabetes mellitus2. Craighead and coworkers observed experimental diabetes in mice after inoculation of encephalomyocarditis virus3. Other viruses suspected of inducing diabetes are coxsackie, mumps, rubella and cytomegalo virus4.
 Bovine viral diarrhea mucosal disease (BVD-MD) is a frequently fatal infectious disease of cattle. There is no concrete evidence to date that BVD-MD virus induces diabetes. In this report, the clinicopathological characteristics of diabetes mellitus in cattle were described, and the relationship between BVD-MD and diabetes mellitus was discussed.

Bovine Islets of Langerhans: Potential Source for Transplantation?

Two methods are described for large-scale isolation of islets from the bovine pancreas. With the single-endpoint technique the average number of islets isolated from a 73.3-g pancreas sample was 150,560, representing an estimated total islet volume of 0.391 ml. Pancreases processed by the continuous digestion-filtration method yielded an average of 246,229 islets from a 93.9-g pancreas sample, representing an estimated total islet volume of 0.458 ml. Isolated bovine pancreatic islets demonstrated a well-preserved endocrine cell ultrastructure, responded to acute stimulation with propionic acid, and temporarily reversed experimental diabetes in mice.

Impaired response of experimental diabetic mice to tricyclics: A possible beta-adrenergic mechanism

Diabetes is reportedly associated with alterations in peripheral and central noradrenergic systems. The latter might be involved in the antidepressant effects of imipramine-like drugs in both humans and animals. Therefore, it is possible that diabetics show an impaired responsiveness to tricyclics. To test this possibility the effects of streptozotocin (STZ)-induced experimental diabetes in mice were assessed in two psychopharmacological tests: 1) the reversal of apomorphine- (16 mg/kg) induced hypothermia and 2) the hypoactivity by a direct beta-agonist (clenbuterol 0.06 mg/kg). At day 15 after STZ or vehicle treatment, imipramine (4 mg/kg) antagonized the apomorphine-induced hypothermia in diabetic (D) and nondiabetic (ND) mice and clenbuterol produced hypoactivity in both groups. At day 30 and 45, the ability of imipramine (1, 2, 4, 8, 16 mg/kg), clomipramine (8 mg/kg) and desipramine (2 mg/kg) to reverse apomorphine-induced hypothermia disappeared at the same time that clenbuterol lost its ability to induce hypomotility in D mice. These impaired responses on both tests were corrected by a short period of insulin therapy. These two tests may reflect central beta- adrenergic functions. Therefore, these data suggest that the impaired responsiveness of diabetic mice might be due at least in part to a noradrenergic dysfunction. Possibly, in diabetes, a beta-adrenoceptor desensitization identical to that observed at the peripheral level occurs in the central nervous system. The possibility that a thyroid hormone deficiency may be involved was also tested. Decreased T3 plasma levels were found in D mice concomitant with the impaired pharmacological responses and T3 supplementation turned these responses to normal. An hypothetical link between these thyroid effects and the beta-adrenergic desensitization in D mice could be suggested but remains to be determined.

Experimental diabetes in mice infected with coxsackie viruses

The influence of Coxsackie B4 and AI3 viruses on the pancreas of mice (resistant and susceptible to diabetes) was studied. Glucose intolerance and changes in the synthesis of immunoreactive insulin were detected in all the treated groups of animals. Biochemical changes were more prominent in male DBA/2 mice, infected with Coxsackie B4 virus, in FI (CBA X C57Bl/6) hybrids and in female DBA/2 mice infected with Coxsackie AI3 virus and alloxan.

Administration of silica or monoclonal antibody to Thy-1 prevents low-dose streptozotocin-induced diabetes in mice

Multiple injections of low doses of streptozotocin induce an experimental diabetes in mice. We have analyzed in two inbred strains whether the development of hyperglycaemia can be influenced by administration of macrophage-toxic silica particles or by a monoclonal antibody to Thy-1.2. Mice received streptozotocin (30 or 40 mg/kg) on five consecutive days (day 0–day 4) and in addition either silica particles (starting at day 0) or anti-Thy-1.2 (starting at day −2 or −3). In both strains mice receiving streptozotocin alone became hyperglycaemic within two weeks. Additional treatment with silica almost fully prevented diabetes development. Anti-Thy-1.2 administration was similarly effective in C57B1/Ks and partially protective in C57BL/6 mice. Histological analysis of pancreatic islets showed that a large fraction of beta cells had been spared from destruction by this treatment. The data indicate a role for both macrophages and Thy-1 positive cells in the pathogenesis of low-dose streptozotocin-induced diabetes.

The influence of the major histocompatibility complex (H-2) on experimental diabetes in mice

Mice with different histocompatibility loci on an identical background genome (congenic resistant lines of mice) were used to study the possible influence of the histocompatibility complex on experimental diabetes. The major histocompatibility complex (H-2) was not found to influence the diabetogenic effect of encephalomyocarditis (EMC) virus. In contrast the glucose intolerance following heterologous and homologous immunization with pancreatic antigens appeared H-2 influenced. Antibodies against cell surface components on viable B-cells were present in serum from mice with glucose intolerance induced by homologous immunization. The results suggest that the susceptibility to experimental autoimmune diabetes in mice is influenced by the H-2 complex.