Systemic Deletion of ARRDC4 Improves Cardiac Reserve and Exercise Capacity in Diabetes.

BACKGROUND: Exercise intolerance is an independent predictor of poor prognosis in diabetes. The underlying mechanism of the association between hyperglycemia and exercise intolerance remains undefined. We recently demonstrated that the interaction between ARRDC4 (arrestin domain-containing protein 4) and GLUT1 (glucose transporter 1) regulates cardiac metabolism. OBJECTIVE: To determine whether this mechanism broadly impacts diabetic complications, we investigated the role of ARRDC4 in the pathogenesis of diabetic cardiac and skeletal myopathy. METHODS AND RESULTS: High glucose promoted translocation of MondoA into the nucleus, which upregulated Arrdc4 transcriptional expression, increased lysosomal GLUT1 trafficking, and blocked glucose transport in cardiomyocytes, forming a feedback mechanism. This role of ARRDC4 was confirmed in human muscular cells from type 2 diabetic patients. Prolonged hyperglycemia upregulated myocardial Arrdc4 expression in multiple types of mouse models of diabetes. We then analyzed hyperglycemia-induced cardiac and skeletal muscle abnormalities in insulin-deficient mice. Hyperglycemia increased advanced glycation end-products and elicited oxidative and endoplasmic reticulum stress leading to apoptosis in the heart and peripheral muscle. However, deletion of Arrdc4 augmented tissue glucose transport and mitochondrial respiration, protecting the heart and muscle from tissue damage. Stress hemodynamic analysis and treadmill exhaustion test uncovered that Arrdc4-knockout mice had greater cardiac inotropic/chronotropic reserve with higher exercise endurance than wild-type (WT) animals under diabetes. While multiple organs were involved in the mechanism, cardiac-specific overexpression (beyond levels observed during diabetes) using adenoassociated virus suggests that high levels of myocardial ARRDC4 have the potential to contribute to exercise intolerance by interfering with cardiac metabolism through its interaction with GLUT1 in diabetes. Importantly, the ARRDC4 mutation mouse line exhibited greater exercise tolerance, showing the potential therapeutic impact on diabetic cardiomyopathy by disrupting the interaction between ARRDC4 and GLUT1. CONCLUSIONS: ARRDC4 serves as a regulator of hyperglycemia-induced toxicities toward cardiac and skeletal muscle, revealing a new molecular framework that connects hyperglycemia to cardiac/skeletal myopathy to exercise intolerance.

Cardiomyocyte-specific Txnip C247S mutation improves left ventricular functional reserve in streptozotocin-induced diabetic mice.

Underlying molecular mechanisms for the development of diabetic cardiomyopathy remain to be determined. Long-term exposure to hyperglycemia causes oxidative stress, which leads to cardiomyocyte dysfunction. Previous studies established the importance of thioredoxin-interacting protein (Txnip) in cellular redox homeostasis and glucose metabolism. Txnip is a highly glucose-responsive molecule that interacts with the catalytic center of reduced thioredoxin and inhibits the antioxidant function of thioredoxin. Here, we show that the molecular interaction between Txnip and thioredoxin plays a pivotal role in the regulation of redox balance in the diabetic myocardium. High glucose increased Txnip expression, decreased thioredoxin activities, and caused oxidative stress in cells. The Txnip-thioredoxin complex was detected in cells with overexpressing wild-type Txnip but not Txnip cysteine 247 to serine (C247S) mutant that disrupts the intermolecular disulfide bridge. Then, diabetes was induced in cardiomyocyte-specific Txnip C247S knock-in mice and their littermate control animals by injections of streptozotocin (STZ). Prolonged hyperglycemia upregulated myocardial Txnip expression in both genotypes. The absence of Txnip's inhibition of thioredoxin in Txnip C247S mutant hearts promoted mitochondrial antioxidative capacities in cardiomyocytes, thereby protecting the heart from oxidative damage by diabetes. Stress hemodynamic analysis uncovered that Txnip C247S knock-in hearts have a greater left ventricular contractile reserve than wild-type hearts under STZ-induced diabetic conditions. These results provide novel evidence that Txnip serves as a regulator of hyperglycemia-induced cardiomyocyte toxicities through direct inhibition of thioredoxin and identify the single cysteine residue in Txnip as a therapeutic target for diabetic injuries.NEW & NORTEWORTHY Thioredoxin-interacting protein (Txnip) has been of great interest as a molecular mechanism to mediate diabetic organ damage. Here, we provide novel evidence that a single mutation of Txnip confers a defense mechanism against myocardial oxidative stress in streptozotocin-induced diabetic mice. The results demonstrate the importance of Txnip as a cysteine-containing redox protein that regulates antioxidant thioredoxin via disulfide bond-switching mechanism and identify the cysteine in Txnip as a therapeutic target for diabetic cardiomyopathy.

Preventing relapse to smoking with transcranial magnetic stimulation: Feasibility and potential efficacy.

Many smokers attempt to quit every year, but 90% relapse within 12 months. Converging evidence suggests relapse is associated with insufficient activation of the prefrontal cortex. Delay discounting rate reflects relative activity in brain regions associated with relapse. High-frequency repetitive transcranial magnetic stimulation (rTMS) of the left dorsolateral prefrontal cortex (LDLPFC) increases cortical excitability and reduces delay discounting rates, but little is known about feasibility, tolerability, and potential efficacy for smoking cessation. We hypothesized that 8 sessions of 20Hz rTMS of the LDLPFC combined with an evidence-based self-help intervention will demonstrate feasibility, tolerability, and potential efficacy in a limited double-blind randomized control trial. Smokers (n=29), abstinent for 24h, motivated to quit, and not using cessation medications, were randomized to active 20Hz rTMS at 110% of Motor Threshold or sham stimulation that replicated the look and sound of active stimulation. Stimulation site was located using the 6cm rule and neuro-navigation. Multiple clinical, feasibility, tolerability, and efficacy measures were examined. Active rTMS decreased delay discounting of $100 (F (1, 25.3694)=4.14, p=.05) and $1000 (F (1, 25.169)=8.42, p<.01), reduced the relative risk of relapse 3-fold (RR 0.29, CI 0.10-0.76, Likelihood ratio χ2 with 1 df=6.40, p=.01), increased abstinence rates (active 50% vs. sham 15.4%, Χ2 (df=1)=3.80, p=.05), and increased uptake of the self-help intervention. Clinical, feasibility, and tolerability assessments were favorable. Combining 20Hz rTMS of the LDLPFC with an evidence-based self-help intervention is feasible, well-tolerated, and demonstrates potential efficacy.

Estrogen-modulated frontal cortical CaMKII activity and behavioral supersensitization induced by prolonged cocaine treatment in female rats.

RATIONALE: Females have been demonstrated repeatedly to be more sensitive to cocaine. The role of the frontal cortex (FCX) in mediating behavioral sensitization and the underlying signaling pathways are unclear. OBJECTIVE: The study was designed to characterize the role of FCX calcium/calmodulin-dependent protein kinase II (CaMKII) activity in the behavioral supersensitization observed in female rats after prolonged cocaine exposure. MATERIALS AND METHODS: Intact female rats that received cocaine for 9 days followed by 7 days of drug withdrawal constituted the model used for studying the mechanism of supersensitization. RESULTS: This cocaine withdrawal treatment resulted in behavioral supersensitization in intact female rats as indicated by an enhanced behavioral response to cocaine challenge assessed on day 16 (7-day withdrawal) and compared to the response on day 9 of cocaine treatment. This treatment regimen did not lead to supersensitization in male or in ovariectomized (OVX) rats. Administration of estrogen to OVX rats restored behavioral supersensitivity to repeated cocaine. FCX CaMKII activity was significantly altered by cocaine in females, and this effect was related to estrogen's presence; cocaine-induced changes in striatal CaMKII activity were, however, less estrogen-sensitive. Furthermore, estrogen-modulated FCX CaMKII activity in cocaine-supersensitized rats was dependent on D(1) dopamine receptor activation. CONCLUSION: Estrogen-modulated D(1) dopamine receptor activity mediates the effects of prolonged cocaine exposure on FCX CaMKII, and this, in turn, may contribute to the development of behavioral supersensitivity to repeated cocaine treatment in intact female rats.

Regulation of cyclin-dependent kinase 5 and calcium/calmodulin-dependent protein kinase II by phosphatidylinositol-linked dopamine receptor in rat brain.

A brain dopamine receptor that modulates phosphatidylinositol (PI) metabolism via the activation of phospholipase Cbeta (PLCbeta) has been described previously. The present study aims to define the downstream signaling cascade initiated by the PI-linked dopamine receptor. Incubation of rat brain frontal cortical slices with 6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF83959), a recently identified selective agonist of the PI-linked D1-like dopamine receptor, elicited transient time- and dose-dependent stimulations of cyclin-dependent kinase 5 (cdk5) and calcium/calmodulin-dependent protein kinase II (CaMK II) activities. The stimulation of these kinases is blocked by 20 microM R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) or the PLCbeta antagonist 1-[6-[[17beta-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) and is attenuated by the protein kinase inhibitor calphostin C or by the intracellular calcium chelator BAPTA, indicating that SKF83959 stimulates cdk5 and CaMK II activities via a PI-linked D1-like dopamine receptor, and PLCbeta and is dependent on protein kinase C and calcium. Although cdk5 and CaMK II are physically associated in native brain tissue, no change in this association was observed in response to SKF83959 stimulation or to the inhibition of either cdk5 by roscovitine or of CaMK by 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) (KN93), suggesting that SKF83959-mediated stimulation of cdk5 or CaMK II is independent of the other kinase and that the association of the two kinases is not modulated by change of kinase activity. Moreover, we found that cdk5 phosphorylates dopamine and cAMP-regulated phosphoprotein at Thr75, whereas CaMK II is responsible for the activation of cAMP response element-binding protein in response to SKF83959 stimulation. The present data provide the first insight into the signaling mechanism for the PI-linked dopamine receptor. This information, in turn, may help in exploring the functional consequences of stimulation of this brain receptor.

Decrements in per pulse release of norepinephrine, antagonist potentiation of release and presynaptic receptor theory.

In recent decades the theory that amine transmitter release at nerve terminals is routinely regulated through negative feedback systems sensing and responding to the instantaneous perineuronal concentration of previously liberated transmitter has assumed pre-eminence. However, observations indicate a major drop off in per pulse transmitter release when only two or four stimulation pulses are administered, reflecting the unexpectedly prompt operation of feedback inhibition. We explored this quandary in our understanding of control of transmitter release by axonal depolarization versus terminal feedback using isotopic norepinephrine and in vitro slices of rabbit hippocampus. A technique of continuous collection of superfusate over a 30min cycle of stimulation utilizing a wide range of intervals between individual stimulation pulses was used. Following simulation with single pulses even 60s apart or pseudo one pulses 150s apart a marked decline in per pulse release was noted. The deficits in per pulse release were not related to the number of pulse delivered at any time over the course of a 30min stimulation period. The pulse decrements were independent of the activity of neuronal uptake and of superfusion flow rate or even individual pulse duration and frequency. Presynaptic receptor antagonists, potentiated efflux near maximally with the second of only two pulses. Potentiations were independent of pulse number, pulse duration, or frequency. No linkage between perineuronal transmitter concentrations and the antagonist potentiation of release was found. However, the decrements in per pulse release with multiple pulses and the potentiation by alpha presynaptic antagonists occurred under the same test conditions. We conclude that the pulse deficit can be looked at as largely attributable to an enhanced efflux with delivery of the first pulse in a train of pulses, rather then to a pattern of progressively declining efflux linked to increasing extracellular transmitter levels as frequency and pulse number increase.

Rate-independent inhibition of 5-HT release by 5-HT in the somadendritic regions of raphe neurons.

The somadendritic regions of raphe neurons respond to exogenous 5-hydroxytryptamine (5-HT) with an inhibition of spontaneous rate and a consequent reduction in local transmitter release, providing evidence for the operation of negative feedback regulation of spontaneous rate. Experiments were done to determine if a release process for 5-HT might also operate in the somadendritic regions that is independent of negative feedback and rate regulation. Slices of rabbit brain containing medullary or midbrain raphe nuclei, were stimulated in vitro at predetermined frequencies and the efflux of 3H-transmitter determined. The stimulation-induced pattern of transmitter release was independent of frequency, pointing to the absence of feedback. Further, exogenous 5-HT (1 x 10(-6)M) depressed the release of 3H-transmitter, but the inhibition, monitored over a range of frequencies, did not reflect competition with endogenous 5-HT for receptor sites. The antagonist methiothepin (3 x 10(-6)M) attenuated the inhibitions by 5-HT but did not by itself potentiate transmitter release, as expected if feedback inhibition were operative. Labeled transmitter release was antagonized by pretreatment with fluoxetine prior to 3H-HT incubation, and was severely curtailed in a calcium deficient medium, confirming that a neuronally relevant pool of transmitter was involved. It is concluded that serotonergic somadendritic sites contain inhibitory receptors for 5-HT release that operate independently of rate regulation and feedback. These findings could explain how other transmitters, and 5-HT itself (through dendritic release of transmitter), could exert synaptic effects on serotonergic and other neurons without being promptly countermanded by a somadendritic feedback-induced rate correction.