Evidence for altered transport of insulin across the blood–brain barrier in insulin-resistant humans

Eating behavior, body weight regulation, peripheral glucose metabolism, and cognitive function depend on adequate insulin action in the brain, and recent studies in humans suggested that impaired insulin action in the brain emerges upon fat intake, obesity, and genetic variants. As insulin enters into the brain in a receptor-mediated fashion, we hypothesized that whole-body insulin sensitivity might affect the transport of insulin into the brain and contribute to the aversive effect of insulin resistance in the central nervous system. In this study, we aimed to determine the ratio of insulin in the cerebrospinal fluid and serum to whole-body insulin sensitivity. Healthy human subjects participated in an oral glucose tolerance test to determine whole-body insulin sensitivity and underwent lumbar puncture. Blood and CSF concentrations of insulin were significantly correlated. The CSF/serum ratio for insulin was significantly associated with whole body insulin sensitivity with reduced insulin transported into the CSF in insulin-resistant subjects. Together, our data suggest that transport of insulin into the CSF relates to peripheral insulin sensitivity and impairs insulin action in the brain. This underlines the need for sensitizing measures in insulin-resistant subjects.     

Excitation–contraction coupling in human heart failure examined by action potential clamp in rat cardiac myocytes

The effect of the loss of the notch in the human action potential (AP) during heart failure was examined by voltage clamping rat ventricular myocytes with human APs and recording intracellular Ca²⁺ with fluorescent dyes. Loss of the notch resulted in about a 50% reduction in the initial phase of the Ca²⁺ transient due to reduced ability of the l-type Ca²⁺ channel to trigger release. The failing human AP increased non-uniformity of cytosolic Ca²⁺, with some cellular regions failing to elicit Ca²⁺-induced Ca²⁺ release from the sarcoplasmic reticulum. In addition, there was an increase in the occurrence of late Ca²⁺ sparks. Monte-Carlo simulations of spark activation by l-type Ca²⁺ current supported the idea that the decreased synchrony of Ca²⁺ spark production associated with the loss of the notch could be explained by reduced Ca²⁺ influx from open Ca²⁺ channels. We conclude that the notch of the AP is critical for efficient and synchronous EC coupling and that the loss of the notch will reduce the SR Ca²⁺ release in heart failure, without changes in (for example) SR Ca²⁺-ATPase uptake.     

Design–based stereological estimation of the total number of cardiac myocytes in histological sections

Background Counting the total number of cardiac myocytes has not previously been possible in ordinary histological sections using light microscopy (LM) due to difficulties in defining the myocyte borders properly.Aim To describe a method by which the total number of cardiac myocytes is estimated in LM sections using design–based stereology. Materials and methods From formalin–fixed left rat ventricles (LV) isotropic uniformly random sections were cut. The total number of myocyte nuclei per LV was estimated using the optical disector. Two–µm–thick serial paraffin sections were stained with antibodies against cadherin and type IV collagen to visualise the intercalated discs and the myocyte membranes, respectively. Using the physical disector in “local vertical windows” of the serial sections, the average number of nuclei per myocyte was estimated.Results The total number of myocyte nuclei in LV was 34.1 × 10⁶ (0.08) (mean (coefficient of variation)), and the mean number of nuclei per myocyte 1.85 (0.03). Combining these estimates the total number of myocytes in LV was calculated to be 18.5 × 10⁶ (0.09).Conclusions This new method is applicable to a range of experiments focusing on myocyte proliferation and death.     

Synthesis of an enzyme—dependent prodrug and evaluation of its potential for colon targeting

AIM：To synthesize dexamethasone-succinate-dextran(DSD)conjugate and to evaluate the potentiality of DSD for the treatment of inflammatory bowel diseases.METHODS:Dexamethasone was attached to dextran(average molecular weight=70400Dalton)using succinate anhydride in an an hydrous environment catalyzed by 4-dimethylaminopyridine and1,1′-carbornyldiimidazole.The chemical structure of DSDwas identified by UV,IR and NMR,and the in vivo drug release behavior of this prodrug was investigated after oral administration of DNA suspension.RESULTS:The DSD conjugate was obtained in two steps and the content of dexamethasone in DSD was11.28%.The dextran prodrug was stable in rat stomach and smal intestine and negligibly absorbed form these tracts.Fout to nine hours after the oral administration.most of the prodrug(>95%)had moved to the cecum and colon,and was easily hydrolyzed by an endodextranase.Recover of dexamethasone from colon and cecum after administration of DSD conjugate was 6-12folds higher than the recovery after administration of unmodified dexamethasone(t=2.74,P<0.050.The prefernetial release of free dexamethasone in cecum and colon over that in the small intestine was staistically significant(t=2.27,P<0.05).CONCLUSION:The results of this study indicate that dependentconjugates may be useful in selectively delivering glucocorticoids to the colon.     

Prevalence of and risk factors for the development of atopic dermatitis in schoolchildren aged 12–14 in northwest Croatia

BackgroundAtopic dermatitis is a common allergic disorder. A multifactorial background for atopic dermatitis has been suggested, with genetic as well as environmental factors influencing disease development. Our aim was to estimate the prevalence rate and associated factors for atopic dermatitis in northern Croatia using the methods of the internationally standardised ISAAC protocol.MethodsThe study was undertaken among 12–14-year-old schoolchildren. Data were collected using standardised ISAAC written questionnaire Phase One and some selected questions from the ISAAC supplementary questionnaire completed by parents.ResultsA total of 2887 children participated in the study. Estimated lifetime (ever) prevalence rate of atopic dermatitis symptoms was 7.55% and estimated 12-month prevalence rate was 5.75%. The factors found to be associated to the symptoms of atopic dermatitis ever were: positive family atopy, female gender, sleeping on feather pillow and contact with pets after age of seven, and to the symptoms in the past 12 months were: positive family atopy, female gender, sleeping on feather pillow, parasite infestation, and contact with pets in the first year of life.ConclusionsThe results of our study show that northern Croatia is a region with moderate prevalence rates of atopic dermatitis. Following risk factors were family atopy, female gender and sleeping on feather pillow. Because of controversial results of previous studies conducted on the same topic further investigations should be made.     

Prevalence of and secular trends in diagnosed diabetes in Italy: 1980–2013

Background and aims

The aim of this research was to examine the prevalence of diabetes in Italy over a 34-year period.

Placental growth factor-1 and epithelial haemato–retinal barrier breakdown: potential implication in the pathogenesis of diabetic retinopathy

Aims/hypothesis Disruption of the retinal pigment epithelial (RPE) barrier contributes to sub-retinal fluid and retinal oedema as observed in diabetic retinopathy. High placental growth factor (PLGF) vitreous levels have been found in diabetic patients. This work aimed to elucidate the influence of PLGF-1 on a human RPE cell line (ARPE-19) barrier in vitro and on normal rat eyes in vivo. Methods ARPE-19 permeability was measured using transepithelial resistance and inulin flux under stimulation of PLGF-1, vascular endothelial growth factor (VEGF)-E and VEGF 165. Using RT-PCR, we evaluated the effect of hypoxic conditions or insulin on transepithelial resistance and on PLGF-1 and VEGF receptors. The involvement of mitogen-activated protein kinase (MEK, also known as MAPK)/extracellular signal-regulated kinase (ERK, also known as EPHB2) signalling pathways under PLGF-1 stimulation was evaluated by western blot analysis and specific inhibitors. The effect of PLGF-1 on the external haemato–retinal barrier was evaluated after intravitreous injection of PLGF-1 in the rat eye; evaluation was by semi-thin analysis and zonula occludens-1 immunolocalisation on flat-mounted RPE. Results In vitro, PLGF-1 induced a reversible decrease of transepithelial resistance and enhanced tritiated inulin flux. These effects were specifically abolished by an antisense oligonucleotide directed at VEGF receptor 1. Exposure of ARPE-19 cells to hypoxic conditions or to insulin induced an upregulation of PLGF-1 expression along with increased transcellular permeability. The PLGF-1-induced RPE cell permeability involved the MEK signalling pathway. Injection of PLGF-1 in the rat eye vitreous induced an opening of the RPE tight junctions with subsequent sub-retinal fluid accumulation, retinal oedema and cytoplasm translocation of junction proteins. Conclusions/interpretaion Our results indicate that PLGF-1 may be a potential regulation target for the control of diabetic retinal and macular oedema. This work was presented in part at the Association for Research and Vision in Ophthalmology meeting, Fort Lauderdale, FL, USA, in May 2005.     

Ablation of Cav2.3 / E–type voltage–gated calcium channel results in cardiac arrhythmia and altered autonomic control within the murine cardiovascular system

Voltage–gated calcium channels are key components in cardiac electrophysiology. We demonstrate that Ca_v2.3 is expressed in mouse and human heart and that mice lacking the Ca_v2.3 voltage–gated calcium channel exhibit severe alterations in cardiac function. Amplified cDNA fragments from murine heart and single cardiomyocytes reveal the expression of three different Ca_v2.3 splice variants. The ablation of Ca_v2.3 was found to be accompanied by a compensatory upregulation of the Ca_v3.1 T–type calcium channel, while other voltage–gated calcium channels remained unaffected. Telemetric ECG recordings from Ca_v2.3 deficient mice displayed subsidiary escape rhythm, altered atrial activation patterns, atrioventricular conduction disturbances and alteration in QRS–morphology. Furthermore, time domain analysis of heart rate variability (HRV) in Ca_v2.3(–|–) mice exhibited a significant increase in heart rate as well as in the coefficient of variance (CV) compared to control mice. Administration of atropin/propranolol revealed that increased heart rate was due to enhanced sympathetic tonus and that partial decrease of CV in Ca_v2.3(–|–) mice after autonomic block was in accordance with a complete abolishment of 2^nd degree atrioventricular block. However, escape rhythms, atrial activation disturbances and QRS–dysmorphology remained unaffected, indicating that these are intrinsic cardiac features in Ca_v2.3(–|–) mice. We conclude that the expression of Ca_v2.3 is essential for normal impulse generation and conduction in murine heart.     

Characterization of the cardiac succinylome and its role in ischemia–reperfusion injury

Succinylation refers to modification of lysine residues with succinyl groups donated by succinyl-CoA. Sirtuin5 (Sirt5) is a mitochondrial NAD⁺-dependent deacylase that catalyzes the removal of succinyl groups from proteins. Sirt5 and protein succinylation are conserved across species, suggesting functional importance of the modification. Sirt5 loss impacts liver metabolism but the role of succinylation in the heart has not been explored. We combined affinity enrichment with proteomics and mass spectrometry to analyze total succinylated lysine content of mitochondria isolated from WT and Sirt5^−/− mouse hearts. We identified 887 succinylated lysine residues in 184 proteins. 44 peptides (5 proteins) occurred uniquely in WT samples, 289 (46 proteins) in Sirt5^−/− samples, and 554 (133 proteins) were common to both groups. The 46 unique proteins in Sirt5^−/− heart participate in metabolic processes such as fatty acid β-oxidation (Eci2) and branched chain amino acid catabolism, and include respiratory chain proteins (Ndufa7, 12, 13, Dhsa). We performed label-free analysis of the peptides common to WT and Sirt5^−/− hearts. 16 peptides from 9 proteins were significantly increased in Sirt5^−/− by at least 30%. The adenine nucleotide transporter 1 showed the highest increase in succinylation in Sirt5^−/− (108.4 fold). The data indicate that succinylation is widespread in the heart and enriched in metabolic pathways. We examined whether the loss of Sirt5 would impact ischemia–reperfusion (I/R) injury and we found an increase in infarct size in Sirt5^−/− hearts compared to WT littermates (68.5⁺/₋ 1.1% Sirt5^−/− vs 39.6⁺/⁻ 6.8% WT) following 20 min of ischemia and 90-min reperfusion. We further demonstrate that I/R injury in Sirt5^−/− heart is restored to WT levels by pretreatment with dimethyl malonate, a competitive inhibitor of succinate dehydrogenase (SDH), implicating alteration in SDH activity as causative of the injury.     

The p75 neurotrophin receptor appears in plasma in diabetic rats—characterisation of a potential early test for neuropathy

Aims/hypothesis This study tested the premise that immunoreactivity representing the p75 neurotrophin receptor (p75^NTR) appears in plasma of diabetic rats in association with the early stages of neuronal dysfunction or damage. We also examined whether treatment beneficial to neuropathy might reduce the p75^NTR immunoreactivity.Methods Plasma proteins were fractionated by SDS-PAGE and immunoblots exposed to p75^NTR antibody, using receptor protein from cultured PC12 cells as an external standard. Rats were made diabetic with streptozotocin for various periods and exsanguinated. Plasma glucose, HbA₁c and plasma proteins were determined. We also studied plasma samples from diabetic mice lacking the gene coding for p75^NTR, as well as the effect of sciatic nerve crush on healthy male Wistar rats.Results Plasma p75^NTR immunoreactivity began to exceed normal levels at 8 weeks after induction of diabetes, and was significantly raised at 10 (p<0.05) and 12 weeks (p<0.001). Treatment between 8 and 12 weeks with insulin, fidarestat (an aldose reductase inhibitor), nerve growth factor and neurotrophin 3 all normalised the plasma p75^NTR immunoreactivity. Plasma from p75^NTR (–/–) mice contained no such immunoreactivity, though it was present in plasma from wild-type mice. Following nerve crush, p75^NTR immunoreactivity appeared in plasma of non-diabetic mice, indicating that this can be a result of nerve trauma.Conclusions/interpretation These observations suggest that plasma p75^NTR immunoreactivity may serve as an early indicator of neuronal dysfunction or damage in diabetes. The time course of its appearance relates well to that of early neuropathy and its response to interventions that are neuroprotective suggests that it might mirror neurological status.     

Effect of adenosine monophosphate-activated protein kinase–p53–Krüppel-like factor 2a pathway in hyperglycemia-induced cardiac remodeling in adult zebrafish

Aims/IntroductionDiabetic cardiomyopathy is a type of myocardial disease. It causes left ventricular hypertrophy, followed by diastolic and systolic dysfunction, eventually leading to congestive heart failure. However, the underlying mechanism still requires further elucidation.Materials and MethodsA high‐glucose zebrafish model was constructed by administering streptozocin intraperitoneally to enhance the development of cardiomyopathy and then treated with adenosine monophosphate‐activated protein kinase (AMPK) activator. Cardiac structure and function, and protein and gene expression were then analyzed. Cardiomyocytes (CMs) culture in vitro using lentivirus were used for detection of AMPK, p53 and Krüppel‐like factor 2a (klf2a) gene expression.ResultsIn the hyperglycemia group, electrocardiogram findings showed arrhythmia, echocardiography results showed heart enlargement and dysfunction, and many differences, such as increased apoptosis and myocardial fiber loss, were observed. The phospho‐AMPK and klf2a expression were downregulated, and p53 expression was upregulated. Activation of phospho‐AMPK reduced p53 and increased klf2a expression, alleviated apoptosis in CMs and improved cardiac function in the hyperglycemic zebrafish. In vitro knockdown system of AMPK, p53 and klf2a using lentivirus illustrated an increased p53 expression and decreased klf2a expression in CMs by inhibiting AMPK. Repression of p53 and upregulation of klf2a expression were observed, but no changes in the expression of AMPK and its phosphorylated type.ConclusionsIn the model of streptozocin‐induced hyperglycemia zebrafish, the reduction of phosphorylated AMPK increased p53, which led to KLF2a decrease to facilitate apoptosis of CMs, inducing the cardiac remodeling and cardiac dysfunction. These results can be reversed by AMPK activator, which means the AMPK–p53–klf2a pathway might be a potential target for diabetic cardiomyopathy intervention.     

Ablation of triadin causes loss of cardiac Ca2+ release units,impaired excitation–contraction coupling,and cardiac arrhythmias

Heart muscle excitation–contraction (E-C) coupling is governed by Ca²⁺ release units (CRUs) whereby Ca²⁺ influx via L-type Ca²⁺ channels (Cav1.2) triggers Ca²⁺ release from juxtaposed Ca²⁺ release channels (RyR2) located in junctional sarcoplasmic reticulum (jSR). Although studies suggest that the jSR protein triadin anchors cardiac calsequestrin (Casq2) to RyR2, its contribution to E-C coupling remains unclear. Here, we identify the role of triadin using mice with ablation of the Trdn gene (Trdn^−/−). The structure and protein composition of the cardiac CRU is significantly altered in Trdn^−/− hearts. jSR proteins (RyR2, Casq2, junctin, and junctophilin 1 and 2) are significantly reduced in Trdn^−/− hearts, whereas Cav1.2 and SERCA2a remain unchanged. Electron microscopy shows fragmentation and an overall 50% reduction in the contacts between jSR and T-tubules. Immunolabeling experiments show reduced colocalization of Cav1.2 with RyR2 and substantial Casq2 labeling outside of the jSR in Trdn^−/− myocytes. CRU function is impaired in Trdn^−/− myocytes, with reduced SR Ca²⁺ release and impaired negative feedback of SR Ca²⁺ release on Cav1.2 Ca²⁺ currents (I_Ca). Uninhibited Ca²⁺ influx via I_Ca likely contributes to Ca²⁺ overload and results in spontaneous SR Ca²⁺ releases upon β-adrenergic receptor stimulation with isoproterenol in Trdn^−/− myocytes, and ventricular arrhythmias in Trdn^−/− mice. We conclude that triadin is critically important for maintaining the structural and functional integrity of the cardiac CRU; triadin loss and the resulting alterations in CRU structure and protein composition impairs E-C coupling and renders hearts susceptible to ventricular arrhythmias.     

Inhibition of Na+–H+ exchange before resuscitation following hemorrhagic shock is cardioprotective in rats

Background

Stimulation of the Na⁺–H⁺ exchanger during resuscitation following hemorrhagic shock results in myocardial injury and dysfunction. Inhibition of the Na⁺–H⁺ exchanger appears to be a new pharmacological tool for myocardial protection following ischemia–reperfusion. Our lab showed that inhibition of the Na⁺–H⁺ exchanger, using amiloride and dimethyl amiloride, before ex vivo resuscitation of isolated perfused hearts protected the myocardium and improved the post-resuscitation myocardial function. The purpose of the present study was to examine the myocardial protective effects of treating the hemorrhagic shocked rats by intra-arterial injection of 20 μM dimethyl amiloride (DMA), a specific Na⁺–H⁺ exchanger blocker, before in vivo resuscitation.

Methods

Sprague–Dawley rats were assigned to hemorrhagic treated or untreated groups (n = 4 per group). After 60 min of hemorrhagic shock, rats were treated or not by injection of 20 μM 5-(N,N-dimethyl)-amiloride (DMA) intra-arterially. Rats were then resuscitated in vivo and monitored for 30 min. Then hearts were harvested and perfused in the Langendorff system for 60 min for measurements of hemodynamic function.

Results

Administration of DMA before in vivo resuscitation following 60 min of hemorrhagic shock and 30 min of in vivo resuscitation, 20 μM DMA intra-arterially significantly improved post-resuscitation myocardial function.

Conclusion

Our results suggest that DMA protects the heart against post-resuscitation myocardial injury.