Streptozotocin-treated Diabetic Rats Research Articles

Metabolomic and lipidomic profiling of the spinal cord in type 2 diabetes mellitus rats with painful neuropathy

In this paper we investigated lipid and metabolite changes in diabetic neuropathy, using untargeted lipidomics and metabolomics analyses of the spinal cords from streptozotocin-treated diabetic rats.170 metabolites and 45 lipids were dysregulated in the painful diabetic neuropathy (PDN) phase. Pathway enrichment analysis revealed perturbations in starch and sucrose, tryptophan, pyrimidine, cysteine and methionine, thiamine, tyrosine, and nucleotides. The disturbance of tyrosine, tryptophan, methionine, triacylglycerol, and phosphatidylethanolamine metabolism indicated that pathological mechanisms in the PDN involved energy metabolism, oxidative stress, and neural reparative regeneration. These revelations offered potential biomarkers for PDN and enriched the comprehension of the complex molecular mechanisms characterizing PDN, establishing a solid foundation for subsequent inquiries into neural convalescence and recovery after PDN.

Effect of hydroalcoholic seed extract of Nigella sativa on hepatic and pancreatic factors of Nrf2 and FGF21 in the regulation of insulin transcription factors of MafA and PDX-1 in streptozotocin-treated diabetic rats

IntroductionNigella sativa (N. sativa), one of the most commonly used medicinal herbs with antioxidant properties, increases blood insulin levels and lowers fasting blood sugar. Nuclear Erythroid Factor-Related Factor 2 (Nrf2) and Fibroblast Growth Factor 21 (FGF21) are two antioxidant factors that are increased by oxidative stress and hyperglycemia. The present study investigated how hydroalcoholic extract of N. sativa seed (HENS) increases blood insulin levels, taking into account changes in antioxidant factors and expression of insulin transcription factors.Materials and methodsTwo groups of male diabetic wistar rats were treated orally with HESN at doses of 200 and 400 mg/kg-body weight for one month. Fasting blood sugar (FBS) and insulin were measured using standard kits by photometric and ELISA methods, respectively. The expression levels of the Nrf2, FGF21 and β-Klotho genes as well as the insulin gene-stimulating transcription factors of MafA and PDX-1 were evaluated using real-time PCR. Oxidative stress was assessed by assessing serum total oxidation status (TOS), malondialdehyde (MDA), and total antioxidant capacity (TAC).ResultsHSEN showed a significant reducing effect on FBS and oxidative biomarkers and an increasing effect on serum insulin levels in treated diabetic rats compared to untreated diabetics (P < 0.05). The elevated levels of NRF2 and FGF21 in the liver and pancreas of the diabetic control group were significantly reduced after treatment with both HESN doses (P < 0.05). Following the ameliorative effects of HENS on pancreatic tissue and the reduction of oxidative stress, the expression level of MafA and PDX1 genes approached the level of these factors in healthy rats (P < 0.05).ConclusionThis study showed the therapeutic effects of HENS on diabetic pancreas by reducing oxidative stress and tissue damage, modifying the expression levels of PDX-1 and MafA genes, and regulating insulin secretion and blood glucose levels.

816-P: Bioprinted Allogeneic Islet–Containing Implants Normalize Blood Glucose Control in Diabetic Rat Models without Immune Suppression

Transplantation of cadaveric islets can reverse type 1 diabetes (T1D) but requires life long immunosuppression. Cell encapsulation has potential to overcome this challenge by blocking immune cell access to islet grafts while allowing nutrient exchange, glucose sensing and insulin release. In this study, 3D tissues consisting of patterned, multi-shell fibres with an islet-containing core and immunoprotective shell were manufactured using Aspect Biosystems’ microfluidic 3D bioprinting technology for implantation in diabetic rats. Blood glucose and animal body weight were monitored for several months following surgery. Upon retrieval, bioprinted tissues were assayed for islet cell viability and function by glucose-stimulated insulin secretion (GSIS) , followed by histology and immunohistochemistry (IHC) to analyze foreign body response (FBR) and immune cell infiltration. 3D bioprinted implants containing reaggregated allogeneic rat islets re-established normoglycemia for &amp;gt; 90 days in streptozotocin-treated diabetic immunodeficient rats, and &amp;gt; 30 days in diabetic immunocompetent rats. Post-retrieval, tissue implants demonstrate high viability and functionality, with absence of immune cell penetration through the shell, confirming its immune protective properties. Modification of the outer shell biomaterial to reduce FBR was shown to extend islet tissue implant functionality. This is the first study describing a 3D bioprinted implant composed of a core/shell micro-fibre used to deliver therapeutic islets into diabetic animals. The promising results obtained in rodent models open the door to further testing in larger animal models and, eventually, T1D patients. Disclosure V.Russo: None. S.Getsios: None. T.J.Kieffer: Consultant; Fractyl Health, Inc., Sigilon Therapeutics, Inc., Employee; ViaCyte, Inc., Research Support; Aspect Biosystems. S.Wadsworth: None. R.Jalili: None. Y.Yu: None. R.Agarwal: None. S.Pan: None. N.Hakimi: None. K.Jahan: None. E.M.Wilts: None. S.Ida: None. Funding National Research Council Canada - Industrial Research Assistance Program (#935249) , Canadian International Innovation Program (#938664) , Genome British Columbia (#GEN009) , Stem Cell Network (#FY20/FBP-7 Kieffer) , Mitacs (#IT19764) ,

Angiogenic Hydrogels to Accelerate Early Wound Healing.

Diabetes mellitus affects an increasing proportion of the population, and is projected to double by 2060. Comorbidities contribute to an interrupted healing process which is delayed, prolonged, and associated with increased susceptibility to infection and unresolved inflammation. This leads to chronic nonhealing wounds and potential amputation. Here, the use of a bioactive angiogenic peptide-based hydrogel, SLan, is examined to improve early wound healing in diabetic rats, and its performance is compared to clinically utilized biosynthetic peptide-based materials such as Puramatrix. Streptozotocin-treated diabetic rats underwent 8mm biopsy wounding in their dorsum. Wounds are treated with either Low (1 w%) SLan, High (4 w%) SLan, phosphate buffered saline (PBS), Puramatrix, or K2 (an unfunctionalized nonbioactive control sequentially similar to SLan), covered with Tegaderm, and monitored on for a month; animals are sacrificed for histomorphic analyses and immunostaining. Pharmacokinetic analysis showing no trafficking of peptides from the wound into the circulation. SLan groups show similar wound contraction as control groups (Puramatrix, PBS, and K2), however, showing marked improvement in healing in earlier time points, including increased deposition of new mature blood vessels. Altogether the results suggest this material can be used to "jumpstart" the diabetic wound healing process.

Hypoglycemic and antidyslipidemic potential of Pleurotus ostreatus in streptozotocin-induced diabetic rats

Oyster Mushroom (Pleurotusostreatus), an edible mushroom, is traditionally being used as a curing. However, hypoglycemic as well as antidyslipidemic effects on methanolic and ethyl acetate extracts of oyster mushroom in streptozotocin-treated diabetic rat model was not investigated yet. Here, we investigated that oral administration of 200 mg/kg oyster mushroom for 30 days were estimated through evaluating the fasting blood glucose level and blood lipid profile of diabetic rat. The results from our studies explained that P. ostreatusoyster mushroom considerably decreased blood glucose level initiated after treatment on second week. However, oyster mushroom was significantly reduced in plasma triglycerides (TG), total cholesterol (TC), as well as low-density lipoprotein-cholesterol (LDL-C). Additionally, high density lipoprotein-cholesterol (HDL-C) was enhanced by the treatment of oyster mushroom. Therefore, the current study suggests that P. ostreatus has antihyperglycemic and antidyslipidemic effect on streptozotocin-induce diabetic rat model.

The protective effects of Crocin on testopathy in fat-fed and streptozotocin-treated diabetic rats: An experimental study.

BackgroundMale hypogonadism is associated with type II diabetes mellitus due to testicular dysfunction. Medicinal plants have received considerable attention for the management of diabetes and its complications.ObjectiveThe aim of present study was to evaluate the anti-diabetic and protective influence of Crocin on testopathy in diabetic rats.Materials and MethodsIn this experimental study, type II of diabetes mellitus was induced by high-fat diet and low dose of streptozotocin. Male Wistar rats (8 weeks, 150–200 gr, 18 rats; n= 6 per group) were divided into a control group (standard diet), diabetic group (streptozotocin+high-fat diet), and treatment group (High-fat diet+streptozotocin+Crocin at 20 mg/kg/day, i.p. for 60 days). After 60 days, animals were euthanized, testis and epididymis were dissected, and weights of testes and sperm count were analyzed. Hematoxylin-eosin-stained was done for histopathological examination. Blood samples were collected for the assessment of serum glucose and cholesterol.ResultsHigh-fat diet and streptozotocin significantly increased the serum glucose and cholesterol levels as compared to the control group (p 0. 001). Moreover, there was a significant decrease in the weight of right (p= 0.008) and left testes (p 0. 001) and also the total sperm count (p= 0.023) in the diabetic group compared with the control group. Current results also identified that type II diabetes mellitus induced degeneration in the morphology of seminiferous tubules. Application of Crocin could significantly decrease serum glucose and cholesterol levels (p= 0.003). Furthermore, Crocin treatment significantly increased the weight of the right (p= 0.026) and left (p= 0.014) testes and total sperm count (p= 0.000). Also, Crocin could attenuate the pathological changes of the testes in the treatment group.ConclusionPresent findings concluded that Crocin treatment improved diabetic testopathy and impairment of seminiferous tubules induced by high-fat diet and streptozotocin.

Moderate-intensity 4 mT static magnetic fields prevent bone architectural deterioration and strength reduction by stimulating bone formation in streptozotocin-treated diabetic rats

Type 1 diabetes mellitus (T1DM) has been associated with deterioration of bone microarchitecture and strength, resulting in increased fracture risk. Substantial studies have revealed the capacity of moderate-intensity static magnetic fields (SMF) on promoting osteoblastogenesis in vitro and stimulating bone growth and bone regeneration in vivo, whereas it is unknown whether SMF can resist T1DM-associated osteopenia/osteoporosis. We herein investigated the potential effects of whole-body SMF exposure with 4mT on bone loss in streptozotocin-induced T1DM rats. We found that SMF exposure for 16weeks inhibited architectural deterioration of trabecular bone and cortical bone and mechanical strength reduction in T1DM rats, as evidenced by the MicroCT and 3-point bending findings. Our serum biochemical, bone histomorphometric and PCR results revealed that SMF induced higher serum osteocalcin, mineral apposition rate and osteoblast number of trabecular bone, and higher skeletal osteocalcin, BMP2 and Runx2 gene expression in T1DM rats, whereas SMF showed no significant alteration in serum CTX, skeletal osteoclast number, or osteoclastogenesis-related RANKL-RANK signaling gene expression. Together, our findings suggest that moderate SMF prevented bone architectural deterioration and strength reduction by inhibiting the reduction of bone formation in T1DM rats, and indicate that SMF might become a promising biophysical countermeasure for T1DM-related osteopenia/osteoporosis.

Erratum: Antidiabetic Activities of an LC/MS Fingerprinted Aqueous Extract of Fagonia cretica L. in Preclinical Models.

Diabetes mellitus is a chronic disease and one of the most important public health challenges facing mankind. Fagonia cretica is a medicinal plant used widely in the Punjab in Pakistan. A recent survey has demonstrated that traditional healers and herbalists frequently use this plant to treat diabetes. In the current study, the traditional medicine was prepared as a tea, and the profile of the main metabolites present in the traditional medicine was analysed via LC/MS/MS. The extract was shown to contain a number of phenolic glycosides including quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, kaempferol-3-O-glycoside, kaempferol-3(6′-malonylglucoside), isorhamnetin-3-O-rutinoside, and isorhamnetin 3-(6″-malonylglucoside) in addition to two unidentified sulphonated saponins. The traditional medicine inhibits α-glucosidase in vitro with an IC50 of 4.62 µg/mL. The hypoglycaemic effect of the traditional medicine was evaluated in normoglycaemic and streptozotocin-treated diabetic rats, using glibenclamide as an internal control. The preparation (250 or 500 mg/kg body weight) was administered once a day for 21 consecutive days. The dose of 500 mg/kg was effective in the management of the disease, causing a 45 % decrease in the plasma glucose level at the end of the experimental period. Histological analysis of pancreatic sections confirmed that streptozotocin/nictotinamide treatment caused destruction of pancreatic islet cells, while pancreatic sections from the treatment groups showed that both the extract and glibenclamide partially prevented this deterioration. The mechanism of this protective effect is unclear. However, such a finding suggests that ingestion of the tea could confer additional benefits and should be investigated further.

Antidiabetic Activities of an LC/MS Fingerprinted Aqueous Extract of Fagonia cretica L. in Preclinical Models

Diabetes mellitus is a chronic disease and one of the most important public health challenges facing mankind. Fagonia cretica is a medicinal plant used widely in the Punjab in Pakistan. A recent survey has demonstrated that traditional healers and herbalists frequently use this plant to treat diabetes. In the current study, the traditional medicine was prepared as a tea, and the profile of the main metabolites present in the traditional medicine was analysed via LC/MS/MS. The extract was shown to contain a number of phenolic glycosides including quercetin-3-O-rutinoside, kaempferol-3-O-rutinoside, kaempferol-3-O-glycoside, kaempferol-3(6'-malonylglucoside), isorhamnetin-3-O-rutinoside, and isorhamnetin 3-(6″-malonylglucoside) in addition to two unidentified sulphonated saponins. The traditional medicine inhibits α-glucosidase in vitro with an IC50 of 4.62 µg/mL. The hypoglycaemic effect of the traditional medicine was evaluated in normoglycaemic and streptozotocin-treated diabetic rats, using glibenclamide as an internal control. The preparation (250 or 500 mg/kg body weight) was administered once a day for 21consecutive days. The dose of 500 mg/kg was effective in the management of the disease, causing a 45 % decrease in the plasma glucose level at the end of the experimental period. Histological analysis of pancreatic sections confirmed that streptozotocin/nictotinamide treatment caused destruction of pancreatic islet cells, while pancreatic sections from the treatment groups showed that both the extract and glibenclamide partially prevented this deterioration. The mechanism of this protective effect is unclear. However, such a finding suggests that ingestion of the tea could confer additional benefits and should be investigated further.

A fluorescent homogeneous assay for myeloperoxidase measurement in biological samples. A positive correlation between myeloperoxidase-generated HOCl level and oxidative status in STZ-diabetic rats

Myeloperoxidase (MPO) is a key enzyme derived from leukocytes which is associated with the initiation and progression of many inflammatory diseases. Increased levels of MPO may contribute to cellular dysfunction and tissues injury by producing highly reactive oxidants such as hypochlorous acid (HOCl). Myeloperoxidase-generated HOCl is therefore considered as a relevant biomarker of oxidative stress-related damage and its quantitation is of great importance to the study of disease progression.In this context, the current study describes a rapid, sensitive and homogeneous fluorescence-based method for detecting the MPO chlorination activity in biological samples. This assay utilizes 7-hydroxy-2-oxo-2H-chromene-8-carbaldehyde oxime as a selective probe for HOCl detection, and is adapted to 96-well microplates to allow high-throughput quantitation of active MPO.The ability of the method to monitor HOCl release was further investigated in hyperglycemic streptozotocin-treated diabetic rats. The data proved that the present assay has a reliable performance when quantitating the active MPO in the plasma of diabetic animals, a feature of inflammatory disease found concomitant with an elevation of protein carbonyls levels and lipid peroxidation and which was negatively correlated with the ratio of reduced-to-oxidized glutathione.

Light Control of Insulin Release and Blood Glucose Using an Injectable Photoactivated Depot.

In this work we demonstrate that blood glucose can be controlled remotely through light stimulated release of insulin from an injected cutaneous depot. Human insulin was tethered to an insoluble but injectable polymer via a linker, which was based on the light cleavable di-methoxy nitrophenyl ethyl (DMNPE) group. This material was injected into the skin of streptozotocin-treated diabetic rats. We observed insulin being released into the bloodstream after a 2 min trans-cutaneous irradiation of this site by a compact LED light source. Control animals treated with the same material, but in which light was blocked from the site, showed no release of insulin into the bloodstream. We also demonstrate that additional pulses of light from the light source result in additional pulses of insulin being absorbed into circulation. A significant reduction in blood glucose was then observed. Together, these results demonstrate the feasibility of using light to allow for the continuously variable control of insulin release. This in turn has the potential to allow for the tight control of blood glucose without the invasiveness of insulin pumps and cannulas.

A Comparative Summary on Antioxidant-like Actions of Timolol with Other Antioxidants in Diabetic Cardiomyopathy.

Cellular signaling associated with cardiac β-adrenergic receptors (β-AR) is composed of coupled mechanism among β 1-/β2-AR and Gs proteins with contribution of constitutive β3-AR coupling to Gi proteins. However, down-regulation of β-ARs in the heart under pathological conditions is mediated with a signaling G proteins-included mechanism. Additionally, there are serious conflicting data on this field in literature yet. Although some of these conflictions are generally related with either experimental protocols for different approaches or different animal models. To treat cardiovascular disorders, generally, various types of β-blockers are used while their action mechanisms are not fully known yet. Furthermore, although β-blockers are generally used to block the activated β-ARs, they can be used to scavenge free radicals under oxidative stress. Studies, in whole-system, organ or cellular levels, showed that some β-blockers, including timolol, have protective-actions against increased oxidative stress in diseased heart via ROSscavenging. Additionally, it has been mentioned that some β-blockers nicely prevented the development of heart failure in both experimental and clinical studies by restoring sarcoplasmic reticulum (SR) Ca(2+) release channels, RyR2. Since diabetic cardiomyopathy is recognized due to its diminished responsiveness to β1-AR agonist stimulation in the heart with an up-regulation of β 3-AR, inducing a strong negative inotropic effect on left ventricular function, it has been shown that treatment of streptozotocin-diabetic rats with timolol provided a marked cardio-protection. Importantly, it has been also documented that timolol treatment-dependent cardio-protection in diabetic rats includes basically prevention of RyR2- hyperphosphorylation, which, in turn, block Ca(2+)-leakage from SR via scavenging oxidative agents to control redox-state of cardiomyocytes. This action of timolol in diabetic heart is very similar to other known antioxidants, such as N-acetylcysteine or selenium compounds. In this review article, antioxidant-like action of timolol in diabetic cardiomyopathy was summarized in way of comparison with the benefits obtained with other antioxidants in the similar animal model.

Exogenous Angiotensin I Metabolism in Aorta Isolated from Streptozotocin Treated Diabetic Rats.

Purpose. Products of angiotensin (ANG) I metabolism may predispose to vascular complications of diabetes mellitus. Methods. Diabetes was induced with streptozotocin (75 mg/kg i.p.). Rat aorta fragments, isolated 4 weeks later, were pretreated with perindoprilat (3 μM), thiorphan (3 μM), or vehicle and incubated for 15 minutes with ANG I (1 μM). Products of ANG I metabolism through classical (ANG II, ANG III, and ANG IV) and alternative (ANG (1–9), ANG (1–7), and ANG (1–5)) pathways were measured in the buffer, using liquid chromatography-mass spectrometry. Results. Incubation with ANG I resulted in higher concentration of ANG II (P = 0.02, vehicle pretreatment) and lower of ANG (1–9) (P = 0.048, perindoprilat pretreatment) in diabetes. Preference for the classical pathway is suggested by higher ANG III/ANG (1–7) ratios in vehicle (P = 0.03), perindoprilat (P = 0.02), and thiorphan pretreated (P = 0.02) diabetic rat. Within the classical pathway, ratios of ANG IV/ANG II (P = 0.01) and of ANG IV/ANG III (P = 0.049), but not of ANG III/ANG II are lower in diabetes. Conclusions. Diabetes in rats led to preference toward deleterious (ANG II, ANG III) over protective (ANG IV, ANG (1–9), and ANG (1–7)) ANG I metabolites.

Fluorescence dilution technique for measurement of albumin reflection coefficient in isolated glomeruli.

This study describes a high-throughput fluorescence dilution technique to measure the albumin reflection coefficient (σAlb) of isolated glomeruli. Rats were injected with FITC-dextran 250 (75 mg/kg), and the glomeruli were isolated in a 6% BSA solution. Changes in the fluorescence of the glomerulus due to water influx in response to an imposed oncotic gradient was used to determine σAlb. Adjustment of the albumin concentration of the bath from 6 to 5, 4, 3, and 2% produced a 10, 25, 35, and 50% decrease in the fluorescence of the glomeruli. Pretreatment of glomeruli with protamine sulfate (2 mg/ml) or TGF-β1 (10 ng/ml) decreased σAlb from 1 to 0.54 and 0.48, respectively. Water and solute movement were modeled using Kedem-Katchalsky equations, and the measured responses closely fit the predicted behavior, indicating that loss of albumin by solvent drag or diffusion is negligible compared with the movement of water. We also found that σAlb was reduced by 17% in fawn hooded hypertensive rats, 33% in hypertensive Dahl salt-sensitive (SS) rats, 26% in streptozotocin-treated diabetic Dahl SS rats, and 21% in 6-mo old type II diabetic nephropathy rats relative to control Sprague-Dawley rats. The changes in glomerular permeability to albumin were correlated with the degree of proteinuria in these strains. These findings indicate that the fluorescence dilution technique can be used to measure σAlb in populations of isolated glomeruli and provides a means to assess the development of glomerular injury in hypertensive and diabetic models.

11β-HSD1 modulates LPS-induced innate immune responses in adipocytes by altering expression of PTEN.

Inhibition of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) represents a therapeutic target for treating hyperglycemia in type 2 diabetes. Here, we investigate the effects of 11β-HSD1 on the innate immune response of adipocytes to produce proinflammatory cytokines. The 11β-HSD1 inhibitor emodin, or 11β-HSD1-targeted small interfering RNA, dose dependently suppressed IL-6, IL-1β, and TNF-α expression in lipopolysaccharide-treated 3T3-L1 adipocytes. Inhibiting 11β-HSD1 also reduced phosphatase and tensin homologue (PTEN) expression, a negative regulator of phosphatidylinositol 3-kinase effects, whereas 1pM cortisone or dexamethasone induced IL-6 and PTEN levels. PTEN-targeted small interfering RNA decreased IL-6, IL-1β, and TNF-α without affecting 11β-HSD1 levels. Correspondingly, emodin increased phosphorylated protein kinase B (p-PKB) (Ser473) to PKB ratio but not p-PKB (Thr308) to PKB ratio. Emodin did not increase the p-PKB (Ser473) to PKB ratio when the rapamycin-insensitive companion of mTOR was depleted, further supporting the involvement of mammalian target of rapamycin complex 2 in PKB phosphorylation. Moreover, emodin suppressed phosphorylated inhibitor of κB α (p-IκBα) to IκBα ratio and reduced nuclear factor κ B subunit p50 in the nuclear fraction. In contrast, 1pM cortisone or dexamethasone decreased p-PKB (Ser473) to PKB ratio, increased p-IκBα to IκBα ratio, and increased nuclear NF-κB subunit p50. Additionally, wortmannin had similar effects on IL-6, p-PKB (Ser473) to PKB ratio, and p-IκBα to IκBα ratio as 1pM cortisone or dexamethasone. Finally, emodin treatment of streptozotocin diabetic rats on a high-fat diet reduced levels of IL-6, PTEN, Cluster of Differentiation 68, and the ratio of p-IκBα to IκBα in visceral fat, indicating that our findings in vitro may also apply to visceral fat in vivo. Together, these results suggest that inhibiting 11β-HSD1 reduces lipopolysaccharide-induced proinflammatory innate immune responses in adipocytes by down-regulating PTEN expression, leading to activation of the PI3K/PKB pathway.

Association of the TLR4 signaling pathway in the retina of streptozotocin-induced diabetic rats.

Diabetic retinopathy is severe damage to the retina caused by complications of diabetes, and is the prevailing cause of blindness. Accumulating evidence from both animal models and humans suggests that the inflammatory process plays a key role in the development of diabetic retinopathy and is facilitated by innate immune response. The aim of this study was to examine whether the TLR4 signaling pathway was involved in the streptozotocin-induced diabetic rat retina. Diabetes was induced by a single intraperitoneal injection of streptozotocin, and rat diabetic retinopathy was examined at 4 weeks of diabetes duration. Then the accumulated leukocytes were counted in vivo by acridine orange leukocyte fluorography, and the retinal vascular permeability was measured by the Evans blue assay. The expressions of TLR4 and its downstream signaling molecules were measured by RT-PCR or Western blot respectively. To evaluate the effect of blocking TLR4 on diabetic retinopathy, TAK-242, a selective TLR4 antagonist, was administered by intraperitoneal injection. Our results showed that the retina of diabetic rats demonstrated accumulated leukocytes and retinal vascular permeability. The mRNA and protein expressions of TLR4 were upregulated in streptozotocin-treated diabetic rat retina. Furthermore, the protein levels of TLR4 downstream signaling molecules were significantly increased in streptozotocin-treated animals. In addition, the protein levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interferon (IFN)-β, three downstream proinflammatory cytokines of TLR4 signal transduction pathway, were also markedly increased in diabetic rats. Administration of TAK-242 attenuated leukocytes accumulated and retinal vascular permeability, and decreased TLR4 downstream signaling molecules and proinflammatory cytokines in streptozotocin-induced animals. Together, these data have demonstrated that TLR4 has a critical role in streptozotocin-induced diabetic retinopathy at the level of inflammatory cytokine induction, in both the MyD88-dependent and MyD88-independent pathways. TLR4 may become a new potential pharmacological target for treating diabetic retinopathy.

Effects of selective endothelin (ET)-A receptor antagonist versus dual ET-A/B receptor antagonist on hearts of streptozotocin-treated diabetic rats

AimsThe aim was to study the differences in the effectiveness of two types of endothelin (ET) receptor antagonists (selective ET-A or dual ET-A/B antagonists) on the hearts of streptozotocin (STZ)-induced diabetic rats (type I diabetes) at functional and biochemical/molecular levels. Main methodsCitrate saline (vehicle) or STZ was injected into rats. The ET-A/B dual receptor antagonist (SB209670, 1mg/kg/day) and the ET-A receptor antagonist (TA-0201, 1mg/kg/day) were then administered to these rats. One week after injection, the animals were separated into those receiving SB209670, TA-0201 or vehicle by 4-week osmotic mini-pump. Key findingsThe VEGF level and percent fractional shortening in the diabetic heart were significantly decreased compared to the non-diabetic heart, whereas SB209670 and TA-0201 treatments greatly and comparably prevented this decrease. SB209670 treatment was more effective in reversing decreased expressions of KDR and phosphorylated AKT, downstream of VEGF angiogenic signaling, than TA-0201 treatment. The eNOS levels in hearts were significantly higher in diabetic rats than in healthy rats, and this increase was significantly reduced by TA-0210 but not by SB209670 treatment. SignificanceImprovement of KDR mRNA and pAKT levels by SB209670 but not TA-0201 suggests that dual ET-A/-B blockade may be effective in improving intracellular systems of these components in the diabetic rat heart. However, the present study also showed that TA-0201 or SB209670 improved percent fractional shortening and VEGF levels of the diabetic hearts to a similar extent, suggesting that ET-A blockade and dual ET-A/-B blockade are similarly effective in improving cardiac dysfunction in the diabetic rats.

Co-transplantation of endothelial progenitor cells and pancreatic islets to induce long-lasting normoglycemia in streptozotocin-treated diabetic rats.

Graft vascularization is a crucial step to obtain stable normoglycemia in pancreatic islet transplantation. Endothelial progenitor cells (EPCs) contribute to neoangiogenesis and to the revascularization process during ischaemic events and play a key role in the response to pancreatic islet injury. In this work we co-transplanted EPCs and islets in the portal vein of chemically-induced diabetic rats to restore islet vascularization and to improve graft survival. Syngenic islets were transplanted, either alone or with EPCs derived from green fluorescent protein (GFP) transgenic rats, into the portal vein of streptozotocin-induced diabetic rats. Blood glucose levels were monitored and intraperitoneal glucose tolerance tests were performed. Real time-PCR was carried out to evaluate the gene expression of angiogenic factors. Diabetic-induced rats showed long-lasting (6 months) normoglycemia upon co-transplantation of syngenic islets and EPCs. After 3–5 days from transplantation, hyperglycaemic levels dropped to normal values and lasted unmodified as long as they were checked. Further, glucose tolerance tests revealed the animals' ability to produce insulin on-demand as indexed by a prompt response in blood glucose clearance. Graft neovascularization was evaluated by immunohistochemistry: for the first time the measure of endothelial thickness revealed a donor-EPC-related neovascularization supporting viable islets up to six months after transplant. Our results highlight the importance of a newly formed viable vascular network together with pancreatic islets to provide de novo adequate supply in order to obtain enduring normoglycemia and prevent diabetes-related long-term health hazards.

Cardiomyocyte glycophagy is regulated by insulin and exposure to high extracellular glucose

Disturbed systemic glycemic and insulinemic status elicits cardiomyocyte metabolic stress and altered glucose handling. In diabetes, pathological myocardial glycogen accumulation occurs. Recently, evidence of a specific myocardial autophagic degradation pathway for glycogen ("glycophagy") has been reported, differentiated from the more well-characterized protein "macrophagy" pathway. The goal of this study was to identify potential mechanisms involved in cardiac glycogen accumulation, glycophagy, and macrophagy regulation using cultured neonatal rat ventricular myocytes (NRVMs). In NRVMs, insulin-induced Akt phosphorylation was evident with 5 mM-glucose conditions (∼2.3-fold increased). Under high-glucose (30 mM) conditions, insulin-augmented phosphorylation was not observed. Accumulation of glycogen was observed in response to insulin only in high-glucose conditions (∼2-fold increase). Increased expression of the glycophagy marker starch-binding domain-containing protein-1 (STBD1, 25% increase) was observed under high-glucose and insulin conditions. Expression levels of the macrophagy markers p62 and light chain protein 3BII:I were not increased by insulin at either glucose level. Preliminary results from hearts of streptozotocin-treated diabetic rats are supportive of the findings obtained in NRVMs, suggesting diabetes induced elevated expression of STBD1 and of an additional glycophagy marker GABA(A) receptor-associated protein-like 1. Confocal microscopy demonstrated that light chain protein 3B and STBD1 immunomarkers were not colocalized in NRVMs. These findings provide the first evidence that cardiomyocyte glycophagy induction occurs under the influence of insulin and is responsive to extracellular high glucose. This study suggests that the regulation of glycogen content and glycophagy induction in the cardiomyocyte may be linked, and it is speculated that glycogen pathology in diabetic cardiomyopathy has glycophagic involvement.

Zebularine Induces Long-Term Survival of Pancreatic Islet Allotransplants in Streptozotocin Treated Diabetic Rats

BackgroundCoping with the immune rejection of allotransplants or autologous cells in patients with an active sensitization towards their autoantigens and autoimmunity presently necessitates life-long immune suppressive therapy acting on the immune system as a whole, which makes the patients vulnerable to infections and increases their risk of developing cancer. New technologies to induce antigen selective long-lasting immunosuppression or immune tolerance are therefore much needed.Methodology/Principal FindingsThe DNA demethylating agent Zebularine, previously demonstrated to induce expression of the genes for the immunosuppressive enzymes indolamine-2,3-deoxygenase-1 (IDO1) and kynureninase of the kynurenine pathway, is tested for capacity to suppress rejection of allotransplants. Allogeneic pancreatic islets from Lewis rats were transplanted under the kidney capsule of Fischer rats previously made diabetic by a streptozotocin injection (40 mg/kg). One group was treated with Zebularine (225 mg/kg) daily for 14 days from day 6 or 8 after transplantation, and a control group received no further treatment. Survival of the transplants was monitored by blood sugar measurements. Rats, normoglycemic for 90 days after allografting, were subjected to transplant removal by nephrectomy to confirm whether normoglycemia was indeed due to a surviving insulin producing transplant, or alternatively was a result of recovery of pancreatic insulin production in some toxin-treated rats. Of 9 Zebularine treated rats, 4 were still normoglycemic after 90 days and became hyperglycemic after nephrectomy. The mean length of normoglycemia in the Zebularine group was 67±8 days as compared to 14±3 days in 9 controls. Seven rats (2 controls and 5 Zebularine treated) were normoglycemic at 90 days due to pancreatic recovery as demonstrated by failure of nephrectomy to induce hyperglycemia.Conclusions/SignificanceZebularine treatment in vivo induces a long-lasting suppression of the immune destruction of allogeneic pancreatic islets resulting in protection of allograft function for more than 10 weeks after end of treatment.