DM Induction Research Articles

Type-2 diabetic aldehyde dehydrogenase 2 mutant mice (ALDH 2*2) exhibiting heart failure with preserved ejection fraction phenotype can be determined by exercise stress echocardiography.

E487K point mutation of aldehyde dehydrogenase (ALDH) 2 (ALDH2*2) in East Asians intrinsically lowers ALDH2 activity. ALDH2*2 is associated with diabetic cardiomyopathy. Diabetic patients exhibit heart failure of preserved ejection fraction (HFpEF) i.e. while the systolic heart function is preserved in them, they may exhibit diastolic dysfunction, implying a jeopardized myocardial health. Currently, it is challenging to detect cardiac functional deterioration in diabetic mice. Stress echocardiography (echo) in the clinical set-up is a procedure used to measure cardiac reserve and impaired cardiac function in coronary artery diseases. Therefore, we hypothesized that high-fat diet fed type-2 diabetic ALDH2*2 mutant mice exhibit HFpEF which can be measured by cardiac echo stress test methodology. We induced type-2 diabetes in 12-week-old male C57BL/6 and ALDH2*2 mice through a high-fat diet. At the end of 4 months of DM induction, we measured the cardiac function in diabetic and control mice of C57BL/6 and ALDH2*2 genotypes by conscious echo. Subsequently, we imposed exercise stress by allowing the mice to run on the treadmill until exhaustion. Post-stress, we measured their cardiac function again. Only after treadmill running, but not at rest, we found a significant decrease in % fractional shortening and % ejection fraction in ALDH2*2 mice with diabetes compared to C57BL/6 diabetic mice as well as non-diabetic (control) ALDH2*2 mice. The diabetic ALDH2*2 mice also exhibited poor maximal running speed and distance. Our data suggest that high-fat fed diabetic ALDH2*2 mice exhibit HFpEF and treadmill exercise stress echo test is able to determine this HFpEF in the diabetic ALDH2*2 mice.

Improvement in bladder dysfunction after bladder transplantation of amniotic fluid stem cells in diabetic rats

To examine the effects of human amniotic fluid stem cells (hAFSCs) transplantation on bladder function and molecular changes in diabetic rats, 60 female Sprague-Dawley rats were used for study. Three groups were assigned including sham control rats, streptozotocin (STZ, 60 mg/kg)-induced diabetic rats and STZ-induced diabetic rats plus bladder hAFSCs transplantation. Compared to controls, diabetic rats had decreased body weight but increased bladder weight. Cystometries showed non-voiding contraction, residual volume, voided volume and intercontraction interval increased significantly in diabetic rats at week 4 and 12 after DM induction, but improved after hAFSCs transplantation. The immunoreactivities and mRNAs of nerve growth factor (NGF) decreased significantly in diabetic bladder at week 4 and 12 after DM induction, but recovered after hAFSCs transplantation. The immunoreactivities and mRNAs of M2 and M3 muscarinic receptor increased significantly in diabetic bladder at week 4 after DM induction but recovered after hAFSCs transplantation. The immunoreactivity of 8-hydroxy-20-deoxyguanosine increased significantly in diabetic bladder at week 4 and 12 after DM induction but reduced after hAFSCs transplantation. The present study showed bladder dysfunction in STZ-induced diabetic rats could be improved by hAFSCs transplantation into bladder, which may be related to the recovery of bladder NGF and muscarinic receptors.

Previous Exercise Training Reduces Markers of Renal Oxidative Stress and Inflammation in Streptozotocin-Induced Diabetic Female Rats.

The aim of this study is to evaluate the effects of regular moderate exercise training initiated previously or after induction of diabetes mellitus on renal oxidative stress and inflammation in STZ-induced diabetic female rats. For this purpose, Wistar rats were divided into five groups: sedentary control (SC), trained control (TC), sedentary diabetic (SD), trained diabetic (TD), and previously trained diabetic (PTD). Only the PTD group was submitted to treadmill running for 4 weeks previously to DM induction with streptozotocin (40 mg/kg, i.v). After confirming diabetes, the PTD, TD, and TC groups were submitted to eight weeks of exercise training. At the end of the training protocol, we evaluated the following: glycosuria, body weight gain, plasma, renal and urinary levels of nitric oxide and thiobarbituric acid reactive substances, renal glutathione, and immunolocalization of lymphocytes, macrophages, and nuclear factor-kappa B (NF-κB/p65) in the renal cortex. The results showed that exercise training reduced glycosuria, renal TBARS levels, and the number of immune cells in the renal tissue of the TD and PTD groups. Of note, only previous exercise increased weight gain and urinary/renal NO levels and reduced NF-κB (p65) immunostaining in the renal cortex of the PTD group. In conclusion, our study shows that exercise training, especially when initiated previously to diabetes induction, promotes protective effects in diabetic kidney by reduction of renal oxidative stress and inflammation markers in female Wistar rats.

Modifications of expression of genes and proteins involved in DNA repair and nitric oxide metabolism by carbatonides [disodium-2,6-dimethyl-1,4-dihydropyridine- 3,5-bis(carbonyloxyacetate) derivatives] in intact and diabetic rats.

Studies on the pathogenesis of diabetes mellitus complications indicate that the compounds reducing free radicals and enhancing DNA repair could be prospective as possible remedies. Carbatonides, the disodium-2,6-dimethyl-1,4- dihydropyridine-3,5-bis(carbonyloxyacetate) derivatives, were tested for these properties. EPR spectroscopy showed that metcarbatone was an effective scavenger of hydroxyl radicals produced in the Fenton reaction, etcarbatone, and propcarbatone were less effective, styrylcarbatone was ineffective. UV/VIS spectroscopy revealed that styrylcarbatone manifested a hyperchromic effect when interacting with DNA, while all other carbatonides showeda hypochromic effect. Rats with streptozotocin induced type 1 DM were treated with metcarbatone, etcarbatone or styrylcarbatone (all compounds at doses 0.05 mg kg-1 or 0.5 mg kg-1) nine days after the DM approval. Gene expression levels in kidneys and blood were evaluated by quantitative RT-PCR; protein expression - immunohistochemically in kidneys, heart, sciatic nerve, and eyes; DNA breakage - by comet assay in nucleated blood cells. Induction of DM induced DNA breaks; metcarbatone and styrylcarbatone (low dose) alleviated this effect. Metcarbatone and etcarbatone up-regulated mRNA and protein of eNOS in kidneys of diabetic animals; etcarbatone also in myocardium. Etcarbatone reduced the expression of increased iNOS protein in myocardium, nerve, and kidneys. iNos gene expression was up-regulated in kidneys by etcarbatone and metcarbatone in diabetic animals. In blood, development of DM increased iNos gene expression; etcarbatone and metcarbatone normalised it. Etcarbatone up-regulated the expression of H2AX in kidneys of diabetic animals but decreased the production of c-PARP1. Taken together, our data indicate that carbatonides might have a potential as drugs intended to treat DM complications.

Pre-clinical investigation of Diabetes Mellitus as a risk factor for schizophrenia

This study investigated the behavioral and biochemical parameters of DM1 as a risk factor in an animal model of schizophrenia (SZ). All groups: 1 Control (saline+saline); 2 Alloxan (alloxan+saline); 3 Ketamine (saline+ketamine); 4 (Alloxan+Ketamine) were fasted for a period of 18h before the subsequent induction of DM via a single intraperitoneal (i.p) injection of alloxan (150mg/kg). From the 4th to the 10th days, the animals were injected i.p with ketamine (25mg/kg) or saline, once a day, to induce a model of SZ and 30min after the last administration were subjected to behavioral testing. After, the animals were decapitated and the brain structures were removed. Ketamine induced hyperactivity and in the social interaction, ketamine, alloxan and the association of alloxan+ketamine increased the latency and decreased the number of contacts between animals. The animals from the ketamine, alloxan and alloxan+ketamine groups showed a prepulse startle reflex (PPI) deficit at the three intensities (65, 70 and 75dB). Ketamine was shown to be capable of increasing the activity of acetylcholinesterase (AChE) in the brain structures. Combination of alloxan+ketamine seems to have an exacerbated effect within the cholinergic system. For lipid peroxidation and protein carbonyls, alloxan+ketamine appear to have intensified lipid and protein damage in the three structures. Ketamine and the combination of ketamine+alloxan induced DNA damage in both frequency and damage index. This research found a relationship between DM1 and SZ.

Effects of Quercetin and Omega-3 Combination on Nuclear Factor Kappa B (NFκB) Expression Level in Pancreatic Tissue of Rats with Type-2 Diabetes Mellitus

Background: Hyperglycemia increases nuclear factor kappa B (NFκB) expression and promotes cellular injury. Quercetin and omega-3 are expected to regulate NFκB expression. This study aims to measure the effect of combination therapy with quercetin and omega-3 in lowering the expression of NFκB in the pancreatic tissue of rats with type-2 DM as compared to those treated with monotherapy with either agent. Methods: This experimental study involved the use of a paraffin block of pancreatic tissue from 24 male Wistar rats aged 3 months, weighing between 250 g and 350 g. All rats underwent induction of type-2 DM and were divided into 4 groups: K1 (treated daily with placebo), K2 (treated with quercetin at 20 mg·kgBW-1·d-1), K3 (treated with omega-3 at 100 mg·kgBW-1·d-1), and K4 (treated with quercetin at 20 mg·kgBW-1·d-1 and omega-3 at 100 mg·kgBW-1·d-1). Treatments were administered orally for four weeks. Once the treatment was completed, samples of pancreatic tissue were collected for the measurement of the percentage of NFκB expression using immunohistochemical (IHC) staining. Results:The average level of NFκB expression in the pancreatic nuclei of DM rats treated with the combination of omega-3 and quercetin was significantly lower than that of those treated with placebo, quercetin only, or omega-3 only (p < 0.05). Conclusion: The combination of quercetin at 20 mg·kgBW-1·d-1 and omega-3 at 100 mg·kgBW-1·d-1 is significantly more effective in lowering the percentage of NFκB in pancreatic nuclei than monotherapy with either agent.

Diabetes mellitus influences the expression of NPY and VEGF in neurons of rat trigeminal ganglion

BackgroundDiabetes mellitus (DM) influences the trigeminal nerve function by changing the pain response and transduction of the orofacial sensory pathways. It affects the inflammatory response via neuropeptide Y (NPY) and vascular endothelial growth factor (VEGF), which could potentially have a relevant role in the pathophysiology of diabetic neuropathy. The aim was to investigate expression of VEGF and NPY in subpopulations of trigeminal ganglion (TG) neurons in rat models of early DM1 and DM2. MethodsDM1 model was induced by an intraperitoneal (i.p.) injection of streptozotocin (STZ) (55mg/kg). DM2 rats were fed with a high fat diet (HFD) for two weeks and then received 35mg/kg of STZ i.p. Two weeks and 2months after the STZ-diabetes induction, rats were sacrificed and TG was immunohistochemically analyzed for detection of VEGF and NPY expression, and also double immunofluorescence labeling with isolectin (IB4) was completed. ResultsAn increased percentage of NPY+ neurons was observed 2weeks after DM1 and 2months post DM2 induction. NPY immunoreactivity was restricted to IB4-negative small-diameter and IB4+ neurons. Two weeks post induction, DM1 rats showed an increased percentage of VEGF/IB4− large neurons and DM2 rats showed an increased percentage of VEGF/IB4+ neurons. Two months after DM induction, the DM1 group showed a reduced percentage of VEGF/IB4− small neurons. ConclusionThe observed changes may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory trigeminal neurons. The results contribute to the understanding of the basic pathophysiology of trigeminal diabetic neuropathy.

Influence of intensity of exercise training on physical performance and myocardial morphology of female rats with type I diabetes mellitus

Materials and methods Ninety-day-old female Wistar rats were divided into three groups: exercised-control (EC; n=5), high intensity exercised-diabetic (HIED=high intensity training, 80% of the maximum speed in the stress test; n=4) and low intensity exercised-diabetic (LIED=low Intensity training, 40% of the maximum speed in the stress test; n=6). The diabetes was induced in the rats by administration of Alloxan monohydrate Sigma (ALX, 50 mg kg-1 BW). Fatigue strength test and the maximal exercise test were performed before DM induction. Fatigue strength test consisted of treadmill running at 20m/min with slope equal to 0° until the animal could not run spontaneously. Time and distance were determined at the end of the race. Maximal exercise test consisted of treadmill running with a load of 5m /min every 3 min until the animal could not run spontaneously, when it was determined the maximum load. Animals ran on a treadmill running 1 hour/day, 5 days/week for 6 weeks with a load (high or low intensity). After this period animals were sacrificed and hearts removed, weighed and prepared for histomorphometric analysis.

Early systemic microvascular damage in pigs with atherogenic diabetes mellitus coincides with renal angiopoietin dysbalance.

BackgroundDiabetes mellitus (DM) is associated with a range of microvascular complications including diabetic nephropathy (DN). Microvascular abnormalities in the kidneys are common histopathologic findings in DN, which represent one manifestation of ongoing systemic microvascular damage. Recently, sidestream dark-field (SDF) imaging has emerged as a noninvasive tool that enables one to visualize the microcirculation. In this study, we investigated whether changes in the systemic microvasculature induced by DM and an atherogenic diet correlated spatiotemporally with renal damage.MethodsAtherosclerotic lesion development was triggered in streptozotocin-induced DM pigs (140 mg/kg body weight) by administering an atherogenic diet for approximately 11 months. Fifteen months following induction of DM, microvascular morphology was visualized in control pigs (n = 7), non-diabetic pigs fed an atherogenic diet (ATH, n = 5), and DM pigs fed an atherogenic diet (DM+ATH, n = 5) using SDF imaging of oral mucosal tissue. Subsequently, kidneys were harvested from anethesized pigs and the expression levels of well-established markers for microvascular integrity, such as Angiopoietin-1 (Angpt1) and Angiopoietin-2 (Angpt2) were determined immunohistochemically, while endothelial cell (EC) abundance was determined by immunostaining for von Willebrand factor (vWF).ResultsOur study revealed an increase in the capillary tortuosity index in DM+ATH pigs (2.31±0.17) as compared to the control groups (Controls 0.89±0.08 and ATH 1.55±0.11; p<0.05). Kidney biopsies showed marked glomerular lesions consisting of mesangial expansion and podocyte lesions. Furthermore, we observed a disturbed Angpt2/ Angpt1balance in the cortex of the kidney, as evidenced by increased expression of Angpt2 in DM+ATH pigs as compared to Control pigs (p<0.05).ConclusionIn the setting of DM, atherogenesis leads to the augmentation of mucosal capillary tortuosity, indicative of systemic microvascular damage. Concomitantly, a dysbalance in renal angiopoietins was correlated with the development of diabetic nephropathy. As such, our studies strongly suggest that defects in the systemic microvasculature mirror the accumulation of microvascular damage in the kidney.

Effect of Satureja khuzestanica Jamzad extract on serum lipid profile, blood glucose level and body weight gain in diabetes mellitus: a Rattus norvegicus model

The aims of this study were to evaluate the effects of Satureja khuzestanica Jamzad as one of the most important plants in folk medicine on body weight gain, serum glucose levels and lipid profile in male diabetic rats. Sixteen adult male Sprague-Dawley rats were randomly divided into five independent groups (three per group except the positive control group that contains four rats) as negative, positive, excipient, dose 100 and 300 mg/kg of S. khuzestanica Jamzad hydroalcoholic extract groups. Diabetes mellitus was induced by intraperitoneal injection of a single dose of 60 mg/kg body weight of streptozotocin. The serum levels of fasting blood sugar (FBS), triglyceride (TG), cholesterol, low-density lipoprotein (LDL), very-low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) were measured at two time points, 3 days after DM induction and at the end of the 30-day study period. Body weight was recorded using a digital scale at days 0 and 30. Our results demonstrated that although using 100 mg/kg of S. khuzestanica Jamzad extract could significantly decrease LDL, VLDL and TG concentration (p 0.05). In conclusion, our study demonstrates that S. khuzestanica have a lipid-lowering and antihyperglycemic properties in male rats as an animal model for human.

Rapamycin promotes podocyte autophagy and ameliorates renal injury in diabetic mice.

The aim was to explore the effects of rapamycin on autophagy and injury of podocytes in streptozocin (STZ)-induced type 1 diabetic mice, and its role in delaying progression of diabetic nephropathy. In this study, male Balb/c mice were divided into three groups: control (n = 12), STZ-induced diabetic (n = 12), and rapamycin-treated diabetic (DM + Rapa) (n = 12), which received intraperitoneal injection of rapamycin (2 mg/kg/48 h) after induction of DM. Levels of urinary albumin (UA), blood urea nitrogen, serum creatinine, and kidney weight/body weight were measured at week 12. Renal pathologic changes, number of podocytes autophagy, and organelles injury were investigated by PAS staining, transmission electron microscopy, and immunofluorescence staining, respectively. Western blot was performed to determine the expression of LC3 (a podocyte autophagy marker), phosphorylated mammalian target of rapamycin, p-p70S6K, bax, and caspase-3 protein. Podocytes count was evaluated by immunofluorescence staining and Wilms tumor 1 immunohistochemistry, and Western blot of nephrin and podocin. The results indicated that rapamycin could reduce the kidney weight/body weight and UA secretion. It could alleviate podocyte foot process fusion, glomerular basement membrane thickening, and matrix accumulation, and increase the number of autophagosomes, and LC3-expressing podocytes. Down-regulation of bax and caspase-3 protein, and up-regulation of nephrin and podocin protein were observed in the glomeruli of diabetic mice after administration of rapamycin. In conclusion, rapamycin can ameliorate renal injury in diabetic mice by increasing the autophagy activity and inhibition of apoptosis of podocytes.

MP47-11 EFFECTS OF CHRONIC ADMINISTRATION OF PDE5 INHIBITOR COMBINED WITH GLYCEMIC CONTROL ON ERECTILE FUNCTION IN STREPTOZOTOCIN-INDUCED DIABETIC RATS

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research II1 Apr 2014MP47-11 EFFECTS OF CHRONIC ADMINISTRATION OF PDE5 INHIBITOR COMBINED WITH GLYCEMIC CONTROL ON ERECTILE FUNCTION IN STREPTOZOTOCIN-INDUCED DIABETIC RATS Woo Suk Choi, Oh Seong Kwon, Jae-Seung Paick, and Soo Woong Kim Woo Suk ChoiWoo Suk Choi More articles by this author , Oh Seong KwonOh Seong Kwon More articles by this author , Jae-Seung PaickJae-Seung Paick More articles by this author , and Soo Woong KimSoo Woong Kim More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1464AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives Chronic treatment with type 5 phosphodiesterase inhibitors (PDE5I) was proven to be effective in animal model of diabetes-induced erectile dysfunction (DMED). In addition, a recent research studied that strict glycemic control had an effect of restoring DMED. This study evaluated the effect of chronic administration of PDE5I combined with glycemic control on erectile dysfunction that occurs with the lapse of time after induction of DM. Methods Sprague-Dawley rats (8-week old) were divided into five groups (n = 10 in each): normal control (C), diabetes (DM), DM treated with insulin (DM-I), DM treated with PDE5I (DM-P), and DM treated with insulin and PDE5I (DM-I+P). The rats in diabetic groups received an injection of streptozotocin (45mg/kg). After 10 weeks of establishment of diabetes, DM-I were treated with a daily injection of neutral protamine Hagedorn (NPH), and a daily dosage of 20mg/kg of PDE5I (DA-8159) was administered to DM-P for 4 weeks, respectively. DM-I+P were treated with both treatments simultaneously. After 14 weeks of establishment of diabetes, cavernosometry with cavernous nerve electrostimulation was performed. Penile tissues were harvested for immunohistochemical evaluation of apoptosis and endothelial marker (CD31), and immunoblot analysis for phosphorylation of e-NOS, Akt and MYPT. Results The rats in group DM showed markedly lower erectile parameters than those in the group C, whereas the rats in DM-I and DM-P showed intermediate erectile function between the group DM and the group C. The Rats in DM-I+P group showed restored erectile function, comparable to the group C (Figure 1). Comparison of apoptotic index, expression of endothelial marker (CD31), and phosphorylation of e-NOS and Akt displayed similar pattern with the results from cavernosometry (DM < DM-I = DM-P < DM-I+P = C, p<0.05), and distribution of phosphorylated MYPT was in reverse order. Conclusions Chronic administration of PDE5I and glycemic control with insulin show a restoring effect of overt DMED. Combination of both treatments produces a larger effect. Multiple mechanisms including apoptosis, endothelial rehabilitation and Rho kinase pathway are responsible for the additional effects. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e522 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Woo Suk Choi More articles by this author Oh Seong Kwon More articles by this author Jae-Seung Paick More articles by this author Soo Woong Kim More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Kefir administration reduced progression of renal injury in STZ-diabetic rats

D mellitus has become a serious public health problem that affects millions of individuals worldwide. The World Health Organization predicts that 439 million people will have this disease in 2030, and Brazil was listed 5th of 10 countries estimated to have the highest number of people with diabetes, affecting approximately 12.7 million Brazilians in 2030. Kefir is a beverage made from milk that is fermented by a complex mixture of bacteria, including various species of lactobacilli and yeasts. It has been considered a probiotic due to its antioxidant and anti-inflammatory properties. The aim of this study was to investigate the effects of Kefir on the production of nitric oxide and oxidative stress and renal damage in STZinduced diabetic rats. Diabetes was induced in adult male Wistar rats with streptozotocin (45 mg/Kg, iv). The animals received Kefir (K) or its vehicle 1.8 mL/day by gavage, starting from 5th day after induction of DM for 8 weeks. The animals were distributed into 4 groups (n=4 each): control (CTL); control Kefir (CTRK); diabetic (DM) and diabetic Kefir (DMK). Before and after treatment, blood and urine were collected for 24 hours to determine the thiobarbituric acid reactive substances (TBARS), NO, C-reactive protein (CRP), creatinine, urea and proteinuria. The data were processed statistically by one-way ANOVA with post-Newman-Keuls test (P<0.05). After sacrificing the animals, the renal cortex was removed for histology, oxidative stress and NOS evaluation. Comparing to CTL rats, DM rats had shown increased levels of glycemia, plasmatic urea, proteinuria, renal NO, superoxide anion, TBARS, and plasmatic CRP; also had been demonstrated a reduction in urinary urea, creatinine, and NO. However, DMK rats had shown a significant improvement in most of these parameters. Despite of lack of differences observed in the expression of endothelial NO synthase (eNOS), the expression of inducible NO synthase (iNOS) was significantly lower in DMK group when compared to DM rats, as assessed by Western blot analysis. Besides that the DMK group presented a significant reduction of glycogen accumulation within the renal tubules when compared to the DM group. These results indicate that Kefir treatment may contribute to better control of glycemia and oxidative stress, which is associated with the amelioration of renal function, suggesting its use as a non-pharmacological adjuvant to delay the progression of diabetic complications. Thus, Kefir may play a role in slowing the metabolic changes that contribute to DN.

A study of the early changes of the level of calcitonin gene-related Peptide and histopathology of penises of rats with experimentally induced type I diabetes mellitus by streptozocin.

IntroductionDiabetes mellitus (DM) is a multiorgan disease that leads to neurovascular complications that disturb the normal erectile function.AimThe aim of the current work was to study the early changes occurring in the level of calcitonin gene-related peptide (CGRP) and histopathological changes in penile tissues of uncontrolled diabetic rats.Materials and MethodsThis study was carried on 50 adult male Sprague-Dawley rats divided into two main groups: group I (control, n = 10) and group II (diabetic, n = 40). Type I DM was induced by a single intraperitoneal injection of streptozotocin (60 mg/kg). The tissue level of CGRP and histopathological examination of rat penises were assessed at 2, 4, 6, and 8 weeks after induction of DM.ResultsCGRP was higher in the diabetic group at 4, 6, and 8 weeks than in the control group. However, endothelial changes and decreased smooth muscles mass started only 2 weeks after induction of DM.ConclusionDeterioration of histopathological features of the uncontrolled diabetic rats corporeal tissues is time dependent. Furthermore, vascular changes seem to precede the neurological changes. El-Kamshoushi AAM, Abdallah WI, Helal SF, El Azhary NM, and Hassan EM. A study of the early changes of the level of calcitonin gene-related peptide and histopathology of penises of rats with experimentally induced type I diabetes mellitus by streptozocin. Sex Med 2013;1:21–29.

Effects of dual targets intervention on the expression of vascular endothelial growth factor and connective tissue growth factor in diabetic rat retina

Objective To observe the effects of dual targets intervention on the expression of vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) in diabetic rat retina.Methods Forty-eight Sprague-Dawley rats were randomly divided into control group (CON1 group) and diabetes mellitus group (DM group).The rats of DM group were induced with streptozotocin injection creating a diabetic model.Retinas were obtained at eight,10,12 weeks after DM induction from both groups.CTGF and VEGF mRNA levels were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR).Based on the results of above experiments,60 rats with same conditions were selected.Fifty rats were induced with streptozotocin injection creating a diabetic model,and 10 rats comprised the control group (CON2 group).Then the 50 diabetic rats were randomly divided into ranibizumab and CTGF shRNA dual targets intervention group,ranibizumab single-target intervention group,CTGF shRNA singletarget intervention group and non-intervention group.Retinas were obtained at one week after intervention from all the groups.CTGF and VEGF mRNA levels were examined by RT-PCR.Results The levels of CTGF mRNA were significantly higher in DM group than that in CON1 group at the 8th weeks after DM induction,and this up-regulation was maintained through the 12th week (t=-2.49,-2.67,-2.42;P＜0.05).There was no difference on VEGF mRNA levels between DM group and CON1 group at the 8th weeks after DM induction (t=-0.443,P =0.669).VEGF mRNA levels of DM group started to be significantly elevated over those in the CON1 group at the 10th week,and remained to be higher at the 12th week (t=-2.35,-2.57; P＜0.05).The VEGF mRNA of ranibizumab single-target intervention group was significantly lower than that in non-intervention group (t=-3.44,P=0.014),which was similar to CON2 group (t =-1.37,P＞ 0.05) ; however,the CTGF mRNA level was significantly increased as compared to the non-intervention group (t =2.48,P ＜ 0.05).In the CTGF shRNA single-target intervention group,the levels of CTGF and VEGF mRNA were decreased as compared to the nonintervention group (t =0.23,-2.92; P＜ 0.05).In the ranibizumab and CTGF shRNA dual targets intervention group,the levels of CTGF and VEGF mRNA were decreased as compared to the nonintervention group (t=-6.09,-5.11; P＜0.001),which was similar to CON2 group (t=-1.16,1.139; P＞0.05).Conclusions Both CTGF and VEGF gene expression are up-regulated in early diabetic rat retina,and the level of CTGF increased earlier than VEGF.Ranibizumab combined with CTGF shRNA could simultaneously reduce the level of CTGF and VEGF mRNA in diabetic rat retina. Key words: Diabetic retinopathy/therapy; Antibodies, monoclonal/therapeutic use; Vascular endothelial growth factors; Animal experimentation

DOα−β+ expression in favor of HLA-DR engagement in exosomes

The expression of DOβ and not DOα, in addition to the high intracellular DR, low DM levels and absence of surface DR expression in K562 and HL-60 cells introduce alternative regulatory pathways in DR trafficking and consequently the antigen presentation process. The present study attempted to define the naturally occurring DOα negative state and explain the role of DOβ in the intracellular DR accumulation in K562 and HL-60 cells. Despite the absence of DOα, the DOβ chain was detected in the endosomal compartments. The lack of DOα was found to be partially responsible for the absence of DR from the cell membrane since stable K562-DOα transfectants allowed expression of membrane DR. This expression could be significantly increased upon DM induction by IFN-γ, indicating that DM was another limiting factor for the migration of DR to the cell surface of K562 and HL-60 cells. Furthermore, intracellular DR co-localized with the exosome specific marker CD9, while culture supernatants were shown to contain exosome-engaged and exosome free DR activity as evaluated by SDS-page followed by western blot, ELISA and transmission electron microscopy analysis. These findings indicated that in DOα−β+ cells, DR molecules were programmed to secretion rather than surface expression. The presented results provide novel regulatory processes as to DR trafficking, avoiding expression to the cell surface.

Fluvastatin Attenuates Diabetes-Induced Cardiac Sympathetic Neuropathy in Association With a Decrease in Oxidative Stress

Increased oxidative stress might contribute to diabetic (DM) neuropathy, so the effects of long-term treatment with fluvastatin (FL) on myocardial oxidative stress and cardiac sympathetic neural function were investigated in diabetic rats. FL (10 mg . kg(-1) . day(-1), DM-FL) or vehicle (DM-VE) was orally administered for 2 weeks to streptozotocin-induced DM rats. Cardiac oxidative stress was determined by myocardial 8-iso-prostaglandin F(2alpha) (PGF(2alpha)) and NADPH oxidase subunit p22(phox) mRNA expression. Sympathetic neural function was quantified by autoradiography using (131)I- and (125)I-metaiodobenzylguanidine (MIBG). FL did not affect plasma glucose levels but remarkably decreased PGF(2alpha) levels compared with DM-VE rats (13.8+/-9.2 vs 175.0+/-93.9 ng/g tissue), although PGF(2alpha) levels were below the detection limit in non-DM rats. FL significantly reduced myocardial p22(phox) mRNA expression. Cardiac (131)I-MIBG uptake was lower in DM-VE rats than in non-DM rats, but the decrease was attenuated in DM-FL rats (1.31+/-0.08, 1.88+/-0.22, and 1.58+/-0.18 %kg dose/g, respectively, P<0.01). Cardiac MIBG clearance was not affected by the induction of DM or by FL, indicating that the reduced MIBG uptake in DM rats might result from impaired neural function. FL ameliorates cardiac sympathetic neural dysfunction in DM rats in association with attenuation of increased myocardial oxidative stress.

Diabetes reduces bone marrow and circulating porcine endothelial progenitor cells, an effect ameliorated by atorvastatin and independent of cholesterol

Bone marrow derived endothelial progenitor cells (EPCs) are early precursors of mature endothelial cells which replenish aging and damaged endothelial cells. The authors studied a diabetic swine model to determine if induction of DM adversely affects either bone marrow or circulating EPCs and whether a HMG-CoA reductase inhibitor (statin) improves development and recruitment of EPCs in the absence of cholesterol lowering. Streptozotocin was administered to Yorkshire pigs to induce DM. One month after induction, diabetic pigs were treated with atorvastatin (statin, n = 10), ezetimibe (n = 10) or untreated (n = 10) and evaluated for number of bone marrow and circulating EPCs and femoral artery endothelial function. There was no effect of either medication on cholesterol level. One month after induction of DM prior to administration of drugs, the number of bone marrow and circulating EPCs significantly decreased (P < 0.0001) compared to baseline. Three months after DM induction, the mean proportion of circulating EPCs significantly increased in the atorvastatin group, but not in the control or ezetimibe groups. The control group showed progressive reduction in percentage of flow mediated vasodilatation (no dilatation at 3 months) whereas the atorvastatin group and ezetimibe exhibited vasodilatation, 6% and 4% respectively. DM results in significant impairment of bone marrow and circulating EPCs as well as endothelial function. The effect is ameliorated, in part, by atorvastatin independent of its cholesterol lowering effect. These data suggest a model wherein accelerated atherosclerosis seen with DM may, in part, result from reduction in EPCs which may be ameliorated by treatment with a statin.

Abstract 5364: Vitamin B1 Analogue Benfotiamine Supplementation Prevents Diabetic Cardiomyopathy through Activation of eNOS/Akt Signaling Pathway

Cardiomyopathy (DCM) is increasingly recognized as contributing to mortality and morbidity of diabetic patients. Importantly, initial signs of cardiac dysfunction are detected in 75% of young patients with well controlled diabetes, suggesting the necessity for early prevention. Vitamin B1 is defective in diabetics and supplementation with vitamin B1 analogue Benfotiamine (BFT) has been demonstrated to be protective against experimental diabetic limb microangiopathy, nephropathy and retinopathy. Here, we tested whether supplementation of BFT could exhibit its protective effects against DCM. Streptozocin-induced CD1 diabetic (DM) mice were fed with BFT (70mg/kg/day) or vehicle starting from 2wks after glycosuria documentation. Cardiac function was monitored every month using Vevo high resolution echocardiography. 12wks after DM induction, the animals given vehicle started developing the signs of diastolic and systolic dysfunction which was more significant at 20wks of DM. However, BFT (without insulin supplement) markedly suppressed the DM induced left ventricular cavity dilatation (LV end-diastolic volume; 61±4 vs 74±5μl, p&lt;0.001), improved fractional shortening (40±4 vs 26±4%, P&lt;0.001) and ejection fraction (72±5 vs 54±4%, p&lt;0.001) and shifted pressure volume loop leftward compared with the vehicle treated animals. BFT also improved indexes of systolic and diastolic function (dp/dt max 5886±400 vs 4730±356mmHg/s and dp/dt min 3963±282 vs 3145±284mmHg/s, P&lt;0.05 for both comparison). Further, BFT benefited cardiac perfusion (4.5±0.4 vs 3.8±0.1ml/min/gm of tissue, P&lt;0.01, using fluorescent microspheres), which was supported by increased microvasculature (4332±321 vs 3654±263 capillaries/mm 2 ). Moreover, BFT (150μM) reduced the high glucose (30mM) induced apoptosis of cultured adult cardiomyocytes by 21±4% (P&lt;0.05) through activation of the cell survival Akt and eNOS pathway, suggesting its direct effect on cardiomyocytes. Altogether, these results demonstrate that BFT supplementation protects the heart from DM induced cardiac dilatation and dysfunction through activation of prosurvival mechanisms and preservation of myocardial perfusion. Thus, BFT merits attention for potential application in clinical practice.

Dysregulation of cGMP‐dependent protein kinase 1 (PKG‐1) impairs erectile function in diabetic rats: influence of in vivo gene therapy of PKG1α

To investigate the expression of cGMP-dependent protein kinase 1 (PKG1)alpha and PKG1beta in the corpus cavernosum, and to evaluate the effect of adenoviral gene transfer of PKG1alpha to the erectile compartment on erectile function in a rat model of diabetes. Diabetic (DM; induced by streptozotocin) male Sprague Dawley rats were transfected with adenoviruses (AdCMVbetagal or AdCMVPKG1alpha, in 10 rats each) 2 months after the induction of DM. Intracavernosal pressure (ICP) during stimulation of the cavernosal nerve (CN) was assessed, and compared with mean arterial pressure (MAP). Erectile tissue was harvested for Western blot analysis, immunohistochemistry and total PKG activity. Ten age-matched rats without DM served as the control. Compared to controls, AdCMVbetagal-transfected DM rats had significantly lower peak ICP responses, ICP/MAP ratios, and filling rates during CN stimulation. In DM rats transfected with AdCMVPKG1alpha, peak ICP, ICP/MAP ratios and filling rates were significantly better than in DM rats transfected with the reporter gene. As assessed by Western blot and immunohistochemistry, expression of PKG1alpha and PKG1beta was lower in corporal tissue from DM AdCMVbetagal-transfected rats than in controls. PKG1alpha expression was improved after AdCMVPKG1alpha gene therapy. Total PKG activity was lower in DM rat corporal tissue than in controls, and PKG1alpha gene transfer significantly improved DM corporal PKG activity to a value greater than in the control. PKG1alpha and PKG1beta activities are reduced in the erectile tissue of the diabetic rat, and gene transfer of PKG1alpha to the penis restored PKG activity and erectile function in vivo in diabetic rats. Gene therapy procedures targeting PKG1alpha might be an interesting future therapeutic approach to overcome diabetic erectile dysfunction resistant to oral pharmacotherapy.