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An estimated 9% of the American population experiences type II diabetes mellitus (T2DM) due to diet or genetic predisposition. Recent reports indicate that patients with T2DM are at increased risk for cognitive dysfunctions, as observed in conditions like Alzheimer's disease (AD). In addition, AD is the leading cause of dementia, highlighting the urgency of developing novel therapeutic targets for T2DM-induced cognitive deficits. The peroxisome proliferator activated receptor-δ (PPAR-δ) is highly expressed in the brain and has been shown to play an important role in spatial memory and hippocampal neurogenesis. However, the effect of PPAR-δ agonists on T2DM-induced cognitive impairment has not been explored. In this study, the effects of GW0742 (a selective PPAR-δ agonist) on hippocampal synaptic transmission, plasticity, and spatial memory were investigated in the db/db mouse model of T2DM. Oral administration of GW0742 for 2 weeks significantly improved hippocampal long-term potentiation. In addition, GW0742 effectively prevented deficits in hippocampal dependent spatial memory in db/db mice. PPAR-δ-mediated improvements in synaptic plasticity and behavior were accompanied by a significant recovery in hippocampal α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated synaptic transmission. Our findings suggest that activation of PPAR-δ might ameliorate T2DM-induced impairments in hippocampal synaptic plasticity and memory.

Aims Inflammation is considered to be one of the crucial pathological factors associated with the development of Alzheimer's disease, although supportive experimental evidence remains undiscovered. Therefore, the current study was carried out to better understand and establish the pathophysiological involvement of chronic inflammation in a double transgenic mouse model of Alzheimer's disease. Main methods We analyzed amyloid-beta deposition, oxidative stress, biochemical, neurochemical and immunological markers in a 10 month old (APΔE9) mouse model. Memory functions were assessed by behavioral testing followed by measurement of synaptic plasticity via extracellular field recordings. Key findings Substantial increases in amyloid-beta levels, beta-secretase activity, and oxidative stress, along with significant neurochemical alterations in glutamate and GABA levels were detected in the brain of APΔE9 mice. Interestingly, marked elevations of pro-inflammatory cytokines in whole brain lysate of APΔE9 mice were observed. Flow cytometric analysis revealed a higher frequency of CD4 + IL-17a and IFN-γ secreting T-cells in APΔE9 brain, indicating a robust T-cell infiltration and activation. Behavioral deficits in learning and memory tasks, along with impairment in long-term potentiation and associated biochemical changes in the expression of glutamatergic receptor subunits were evident. Significance Thus, this study establishes the role by which oxidative stress, alterations in glutamate and GABA levels and inflammation increases hippocampal and cortical neurotoxicity resulting in the cognitive deficits associated with Alzheimer's disease.

Duchenne Muscular Dystrophy (DMD) is associated with progressive cardiac pathology; however, the SIRT1/PGC1-α activator quercetin may cardioprotect dystrophic hearts. We tested the extent to which long-term 0.2% dietary quercetin enrichment attenuates dystrophic cardiopathology in Mdx/Utrn+/− mice. At 2 mo, Mdx/Utrn+/− mice were fed quercetin-enriched (Mdx/Utrn+/−-Q) or control diet (Mdx/Utrn+/−) for 8 mo. Control C57BL/10 (C57) animals were fed a control diet for 10 mo. Cardiac function was quantified by MRI at 2 and 10 mo. Spontaneous physical activity was quantified during the last week of treatment. At 10 mo hearts were excised for histological and biochemical analysis. Quercetin feeding improved various physiological indexes of cardiac function in diseased animals. Mdx/Utrn+/−-Q also engaged in more high-intensity physical activity than controls. Histological analyses of heart tissues revealed higher expression and colocalization of utrophin and α-sarcoglycan. Lower abundance of fibronectin, cardiac damage (Hematoxylin Eosin-Y), and MMP9 were observed in quercetin-fed vs. control Mdx/Utrn+/− mice. Quercetin evoked higher protein abundance of PGC-1α, cytochrome c, ETC complexes I–V, citrate synthase, SOD2, and GPX compared with control-fed Mdx/Utrn+/−. Quercetin decreased abundance of inflammatory markers including NFκB, TGF-β1, and F4/80 compared with Mdx/Utrn+/−; however, P-NFκB, P-IKBα, IKBα, CD64, and COX2 were similar between groups. Dietary quercetin enrichment improves cardiac function in aged Mdx/Utrn+/− mice and increases mitochondrial protein content and dystrophin glycoprotein complex formation. Histological analyses indicate a marked attenuation in pathological cardiac remodeling and indicate that long-term quercetin consumption benefits the dystrophic heart. NEW & NOTEWORTHY The current investigation provides first-time evidence that quercetin provides physiological cardioprotection against dystrophic pathology and is associated with improved spontaneous physical activity. Secondary findings suggest that quercetin-dependent outcomes are in part due to PGC-1α pathway activation.

Adiponectin, the most abundant plasma adipokine, plays an important role in the regulation of glucose and lipid metabolism. Adiponectin also possesses insulin-sensitizing, anti-inflammatory, angiogenic, and vasodilatory properties which may influence central nervous system (CNS) disorders. Although initially not thought to cross the blood-brain barrier, adiponectin enters the brain through peripheral circulation. In the brain, adiponectin signaling through its receptors, AdipoR1 and AdipoR2, directly influences important brain functions such as energy homeostasis, hippocampal neurogenesis, and synaptic plasticity. Overall, based on its central and peripheral actions, recent evidence indicates that adiponectin has neuroprotective, antiatherogenic, and antidepressant effects. However, these findings are not without controversy as human observational studies report differing correlations between plasma adiponectin levels and incidence of CNS disorders. Despite these controversies, adiponectin is gaining attention as a potential therapeutic target for diverse CNS disorders, such as stroke, Alzheimer’s disease, anxiety, and depression. Evidence regarding the emerging role for adiponectin in these disorders is discussed in the current review.

Honokiol (poly-phenolic lignan from Magnolia grandiflora) is a Sirtuin-3 (SIRT3) activator which exhibit antioxidant activity and augment mitochondrial functions in several experimental models. Modern evidence suggests the critical role of SIRT3 in the progression of several metabolic and neurodegenerative diseases. Amyloid beta (Aβ), the precursor to extracellular senile plaques, accumulates in the brains of patients with Alzheimer's disease (AD) and is related to the development of cognitive impairment and neuronal cell death. Aβ is generated from amyloid-β precursor protein (APP) through sequential cleavages, first by β-secretase and then by γ-secretase. Drugs modulating this pathway are believed to be one of the most promising strategies for AD treatment. In the present study, we found that Honokiol significantly enhanced SIRT3 expression, reduced reactive oxygen species generation and lipid peroxidation, enhanced antioxidant activities, and mitochondrial function thereby reducing Aβ and sAPPβ levels in Chinese Hamster Ovarian (CHO) cells (carrying the amyloid precursor protein-APP and Presenilin PS1 mutation). Mechanistic studies revealed that Honokiol affects neither protein levels of APP nor α-secretase activity. In contrast, Honokiol increased the expression of AMPK, CREB, and PGC-1α, thereby inhibiting β-secretase activity leading to reduced Aβ levels. These results suggest that Honokiol is an activator of SIRT3 capable of improving antioxidant activity, mitochondrial energy regulation, while decreasing Aβ, thereby indicating it to be a lead compound for AD drug development.

Adiponectin has been reported to exert protective effects during pathological ventricular remodeling, but the role of adiponectin in volume overload-induced heart failure remains unclear. In this study we investigated the effect of adiponectin on cardiac myocyte contractile dysfunction following volume overload in rats.Volume overload was surgically induced in rats by infrarenal aorta-vena cava fistula. The rats were intravenously administered adenoviral adiponectin at 2-, 6- and 9-weeks following fistula. The protein expression of adiponectin, adiponectin receptors (AdipoR1/R2 and T-cadherin) and AMPK activity were measured using Western blot analyses. Isolated ventricular myocytes were prepared at 12 weeks post-fistula to examine the contractile performance of myocytes and intracellular Ca(2+) transient.A-V fistula resulted in significant reductions in serum and myocardial adiponectin levels, myocardial adiponectin receptor (AdipoR1/R2 and T-cadherin) levels, as well as myocardial AMPK activity. Consistent with these changes, the isolated myocytes exhibited significant depression in cell shortening and intracellular Ca(2+) transient. Administration of adenoviral adiponectin significantly increased serum adiponectin levels and prevented myocyte contractile dysfunction in fistula rats. Furthermore, pretreatment of isolated myocytes with recombinant adiponectin (2.5 μg/mL) significantly improved their contractile performance in fistula rats, but had no effects in control or adenoviral adiponectin-administered rats.These results demonstrate a positive correlation between adiponectin downregulation and volume overload-induced ventricular remodeling. Adiponectin plays a protective role in volume overload-induced heart failure.

Doxorubicin (DOX) is a highly effective anti-neoplastic agent; however, its cumulative dosing schedules are clinically limited by the development of cardiotoxicity. Previous studies have attributed the cause of DOX-mediated cardiotoxicity to mitochondrial iron accumulation and the ensuing reactive oxygen species (ROS) formation. The present study investigates the role of frataxin (FXN), a mitochondrial iron-sulfur biogenesis protein, and its role in development of DOX-mediated mitochondrial dysfunction. Athymic mice treated with DOX (5 mg/kg, 1 dose/wk with treatments, followed by 2-wk recovery) displayed left ventricular hypertrophy, as observed by impaired cardiac hemodynamic performance parameters. Furthermore, we also observed significant reduction in FXN expression in DOX-treated animals and H9C2 cardiomyoblast cell lines, resulting in increased mitochondrial iron accumulation and the ensuing ROS formation. This observation was paralleled in DOX-treated H9C2 cells by a significant reduction in the mitochondrial bioenergetics, as observed by the reduction of myocardial energy regulation. Surprisingly, similar results were observed in our FXN knockdown stable cell lines constructed by lentiviral technology using short hairpin RNA. To better understand the cardioprotective role of FXN against DOX, we constructed FXN overexpressing cardiomyoblasts, which displayed cardioprotection against mitochondrial iron accumulation, ROS formation, and reduction of mitochondrial bioenergetics. Lastly, our FXN overexpressing cardiomyoblasts were protected from DOX-mediated cardiac hypertrophy. Together, our findings reveal novel insights into the development of DOX-mediated cardiomyopathy.

Interleukin-6 (IL-6) is a pleiotropic cytokine that protects against cardiac ischemia-reperfusion (I/R) injury following pharmacological and ischemic preconditioning (IPC), but the affiliated role in exercise preconditioning is unknown. Our study purpose was to characterize exercise-induced IL-6 cardiac signaling ( aim 1) and evaluate myocardial preconditioning ( aim 2). In aim 1, C57 and IL-6−/− mice underwent 3 days of treadmill exercise for 60 min/day at 18 m/min. Serum, gastrocnemius, and heart were collected preexercise, immediately postxercise, and 30 and 60 min following the final exercise session and analyzed for indexes of IL-6 signaling. For aim 2, a separate cohort of exercise-preconditioned (C57 EX and IL-6−/− EX) and sedentary (C57 SED and IL-6−/− SED) mice received surgical I/R injury (30 min I, 120 min R) or a time-matched sham operation. Ischemic and perfused tissues were examined for necrosis, apoptosis, and autophagy. In aim 1, serum IL-6 and IL-6 receptor (IL-6R), gastrocnemius, and myocardial IL-6R were increased following exercise in C57 mice only. Phosphorylated (p) signal transducer and activator of transcription 3 was increased in gastrocnemius and heart in C57 and IL-6−/− mice postexercise, whereas myocardial iNOS and cyclooxygenase-2 were unchanged in the exercised myocardium. Exercise protected C57 EX mice against I/R-induced arrhythmias and necrosis, whereas arrhythmia score and infarct outcomes were higher in C57 SED, IL-6−/− SED, and IL-6−/− EX mice compared with SH. C57 EX mice expressed increased p-p44/42 MAPK (Thr202/Tyr204) and p-p38 MAPK (Thr180/Tyr182) compared with IL-6−/− EX mice, suggesting pathway involvement in exercise preconditioning. Findings indicate exercise exerts cardioprotection via IL-6 and strongly implicates protective signaling originating from the exercised skeletal muscle.

Background Increased consumption of omega-3 (ω-3) fatty acids found in cold-water fish and fish oil has been reported to protect against obesity. A potential mechanism may be through reduction in adipocyte differentiation. Stearidonic acid (SDA), a plant-based ω-3 fatty acid, has been targeted as a potential surrogate for fish-based fatty acids; however, its role in adipocyte differentiation is unknown. This study was designed to evaluate the effects of SDA on adipocyte differentiation in 3T3-L1 cells. Methods 3T3-L1 preadipocytes were differentiated in the presence of SDA or vehicle-control. Cell viability assay was conducted to determine potential toxicity of SDA. Lipid accumulation was measured by Oil Red O staining and triglyceride (TG) quantification in differentiated 3T3-L1 adipocytes. Adipocyte differentiation was evaluated by adipogenic transcription factors and lipid accumulation gene expression by quantitative real-time polymerase chain reaction (qRT-PCR). Fatty acid analysis was conducted by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Results 3T3-L1 cells treated with SDA were viable at concentrations used for all studies. SDA treatment reduced lipid accumulation in 3T3-L1 adipocytes. This anti-adipogenic effect by SDA was a result of down-regulation of mRNA levels of the adipogenic transcription factors CCAAT/enhancer-binding proteins alpha and beta (C/EBPα, C/EBPβ), peroxisome proliferator-activated receptor gamma (PPARγ), and sterol-regulatory element binding protein-1c (SREBP-1c). SDA treatment resulted in decreased expression of the lipid accumulation genes adipocyte fatty-acid binding protein (AP2), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD-1), lipoprotein lipase (LPL), glucose transporter 4 (GLUT4) and phosphoenolpyruvate carboxykinase (PEPCK). The transcriptional activity of PPARγ was found to be decreased with SDA treatment. SDA treatment led to significant EPA enrichment in 3T3-L1 adipocytes compared to vehicle-control. Conclusion These results demonstrated that SDA can suppress adipocyte differentiation and lipid accumulation in 3T3-L1 cells through down-regulation of adipogenic transcription factors and genes associated with lipid accumulation. This study suggests the use of SDA as a dietary treatment for obesity. Electronic supplementary material The online version of this article (10.1186/s12944-017-0574-7) contains supplementary material, which is available to authorized users.

Elevated blood branched-chain amino acids (BCAA) are often associated with insulin resistance and type 2 diabetes, which might result from a reduced cellular utilization and/or incomplete BCAA oxidation. White adipose tissue (WAT) has become appreciated as a potential player in whole body BCAA metabolism. We tested if expression of the mitochondrial BCAA oxidation checkpoint, branched-chain α-ketoacid dehydrogenase (BCKD) complex, is reduced in obese WAT and regulated by metabolic signals. WAT BCKD protein (E1α subunit) was significantly reduced by 35–50% in various obesity models ( fa/fa rats, db/db mice, diet-induced obese mice), and BCKD component transcripts significantly lower in subcutaneous (SC) adipocytes from obese vs. lean Pima Indians. Treatment of 3T3-L1 adipocytes or mice with peroxisome proliferator-activated receptor-γ agonists increased WAT BCAA catabolism enzyme mRNAs, whereas the nonmetabolizable glucose analog 2-deoxy-d-glucose had the opposite effect. The results support the hypothesis that suboptimal insulin action and/or perturbed metabolic signals in WAT, as would be seen with insulin resistance/type 2 diabetes, could impair WAT BCAA utilization. However, cross-tissue flux studies comparing lean vs. insulin-sensitive or insulin-resistant obese subjects revealed an unexpected negligible uptake of BCAA from human abdominal SC WAT. This suggests that SC WAT may not be an important contributor to blood BCAA phenotypes associated with insulin resistance in the overnight-fasted state. mRNA abundances for BCAA catabolic enzymes were markedly reduced in omental (but not SC) WAT of obese persons with metabolic syndrome compared with weight-matched healthy obese subjects, raising the possibility that visceral WAT contributes to the BCAA metabolic phenotype of metabolically compromised individuals.

Exercise is a potent stimulus against cardiac ischemia reperfusion (IR) injury, although the protective mechanisms are not completely understood. The study purpose was to examine whether the mitochondrial or sarcolemmal ATP-sensitive potassium channel (mito KATPor sarc KATP, respectively) mediates exercise-induced cardioprotection against post-IR cell death and apoptosis. Eighty-six, 4-mo-old male Sprague Dawley rats were randomly assigned to treadmill exercise (Ex; 30 m/min, 3 days, 60 min, ∼70 maximal oxygen uptake) and sedentary (Sed) treatments. Rats were exposed to regional cardiac ischemia (50 min) and reperfusion (120 min) or Sham (170 min; no ligation) surgeries. Exercise subgroups received placebo (saline), 5-hydroxydecanoate (5HD; 10 mg/kg ip), or HMR1098 (10 mg/kg ip) to inhibit mito KATPor sarc KATPchannel. Comprehensive outcome assessments included post-IR ECG arrhythmias, cardiac tissue necrosis, redox perturbations, and autophagy biomarkers. No arrhythmia differences existed between exercised and sedentary hearts following extended-duration IR ( P < 0.05). The sarc KATPchannel was confirmed essential ( P = 0.002) for prevention of antinecrotic tissue death with exercise (percent infarct, Sed = 42%; Ex = 20%; Ex5HD = 16%; ExHMR = 42%), although neither the mito KATP( P = 0.177) nor sarc KATP( P = 0.274) channel provided post-IR protection against apoptosis (terminal deoxynucleotidyl transferase deoxy UTP-mediated nick-end labeling-positive nuclei/mm2, Sham = 1.8 ± 0.5; Sed = 19.4 ± 6.7; Ex = 7.5 ± 4.6; Ex5HD = 14.0 ± 3.9; ExHMR = 11.1 ± 1.8). Exercise preconditioning also appears to preserve basal autophagy levels, as assessed by Beclin 1 ( P ≤ 0.001), microtubule-associated protein-1 light-chain 3B ratios ( P = 0.020), and P62 ( P ≤ 0.001), in the hours immediately following IR. Further research is needed to better understand these findings and corresponding redox changes in exercised hearts.

The nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma plays a key role in regulating whole body glucose homeostasis and insulin sensitivity. Although it is expressed most highly in adipose, it is also present at lower levels in many tissues, including skeletal muscle. The role muscle PPARgamma plays in metabolic regulation and in mediating the antidiabetic effects of the thiazolidinediones is not understood. The goal of this work was to examine the molecular and physiological effects of PPARgamma activation in muscle cells. We found that pharmacological activation of PPARgamma in primary cultured myocytes, and genetic activation of muscle PPARgamma in muscle tissue of transgenic mice, induced the production of adiponectin directly from muscle cells. This muscle-produced adiponectin was functional and capable of stimulating adiponectin signaling in myocytes. In addition, elevated skeletal muscle PPARgamma activity in transgenic mice provided a significant protection from high-fat diet-induced insulin resistance and associated changes in muscle phenotype, including reduced myocyte lipid content and an increase in the proportion of oxidative muscle fiber types. Our findings demonstrate that PPARgamma activation in skeletal muscle can have a significant protective effect on whole body glucose homeostasis and insulin resistance and that myocytes can produce and secrete functional adiponectin in a PPARgamma-dependent manner. We propose that activation of PPARgamma in myocytes induces a local production of adiponectin that acts on muscle tissue to improve insulin sensitivity.

Previous studies have demonstrated the protective signaling of hypoxia-inducible factor (HIF)-1 α against ischemia-reperfusion (I/R) injury in the heart. In the present study, we provide further evidence for a cardioprotective mechanism by HIF-1α against I/R injury exerted via the mitochondrial protein frataxin, which regulates mitochondrial Fe-S cluster formation. Disruption of frataxin has been found to induce mitochondrial iron overload and subsequent ROS production. We observed that frataxin expression was elevated in mice hearts subjected to I/R injury, and this response was blunted in cardiomyocyte-specific HIF-1α knockout (KO) mice. Furthermore, these HIF-1α KO mice sustained extensive cardiac damage from I/R injury compared with control mice. Similarly, reduction of HIF-1α by RNA inhibition resulted in an attenuation of frataxin expression in response to hypoxia in H9C2 cardiomyocytes. Therefore, we postulated that HIF-1α transcriptionally regulates frataxin expression in response to hypoxia and offers a cardioprotective mechanism against ischemic injury. Our promoter activity and chromatin immunoprecipitation assays confirmed the presence of a functional hypoxia response element in the frataxin promoter. Our data also suggest that increased frataxin mitigated mitochondrial iron overload and subsequent ROS production, thus preserving mitochondrial membrane integrity and viability of cardiomyocytes. We postulate that frataxin may exert its beneficial effects by acting as an iron storage protein under hypoxia and subsequently facilitates the maintenance of mitochondrial membrane potential and promotes cell survival. The findings from our study revealed that HIF-1α-frataxin signaling promotes a protective mechanism against hypoxic/ischemic stress.

We examined the effects of interleukin-1β (IL-1β) treatment on the distribution and degradation of lamin B in the nuclear fraction from insulin-secreting RINm5F cells. Western blot analysis indicated that IL-1β treatment caused significant alterations in the redistribution of lamin B, specifically between the Triton X-100-soluble (membrane) and -insoluble (matrix) fractions of the nucleus. IL-1β treatment also increased the lamin carboxymethyltransferase activity and the relative abundance of the carboxymethylated lamin in the nuclear fraction. A significant increase in the relative abundance of lamin B degradation products was also observed in the nuclear fraction from the IL-1β-treated cells. These findings are compatible with a measurable increase in the lamin-degrading caspase-6 activity in IL-1β-treated cells. Confocal microscopic observation of IL-1β-treated cells suggested a significant dissociation of lamin B from the nuclear lamina and its subsequent association with the DNA-rich elements within the nucleus. NG-monomethyl-l-arginine, a known inhibitor of inducible nitric oxide synthetase (iNOS), markedly inhibited IL-1β-induced iNOS gene expression, NO release, caspase-3 and caspase-6 activation, lamin B degradation, and loss of metabolic cell viability, indicating that the observed IL-1β-induced effects on nuclear lamin B involve the intermediacy of NO. Together, our data support the hypothesis that IL-1β treatment results in significant increase in the carboxymethylation of lamin B, which would place lamin B in a strategic location for its degradation mediated by caspases. This could possibly lead to dissolution of the nuclear envelope, culminating in the demise of the effete β-cell.

What is the central question of this study? Does the δ-opioid receptor trigger exercise-induced cardioprotection against ischaemia-reperfusion injury? What is the main finding and its importance? In exercised hearts, the δ-opioid receptor appears to trigger cardioprotection against ischaemia-reperfusion-induced tissue necrosis but not apoptosis.Endogenous opioids mediate exercise-induced cardioprotection against ischaemia-reperfusion (IR) injury, although the opioid receptor subtype mediating this effect is unknown. We investigated whether the δ-opioid receptor mediates exercise-induced cardioprotection against IR injury. Endogenous opioids are produced in various tissues, including the heart and skeletal muscle; therefore, we also sought to identify the effect of exercise on circulating endogenous opioid as well as transcript, protein and receptor expression in heart and skeletal muscle. Male Sprague-Dawley rats (n = 73) were assigned randomly to treadmill exercise or sedentary treatments. Cardiac tissue and serum were harvested 0, 20 and 120 min following exercise and from sedentary animals (n = 32) to quantify effects on proenkephalin and δ-opioid receptor mRNA and protein levels, as well as serum enkephalin. Skeletal muscle (soleus) was harvested at identical time points for determination of proenkephalin protein and mRNA. A separate group of rats (n = 41) were randomly assigned to sham operation (Sham; surgical control), sedentary (Sed), exercise (Ex) or exercise + δ-opioid receptor antagonist (ExD; naltrindole, 5 mg kg(-1) i.p.) and received IR by left anterior descending coronary artery ligation in vivo. After IR, tissues were harvested to quantify treatment effects on necrosis and apoptosis. Cardiac proenkephalin mRNA expression increased following exercise (0 min, P = 0.03; 120 min, P = 0.021), while soleus expression was unaffected. Exercise-induced changes in serum enkephalin were undetectable. After IR, tissue necrosis was elevated in Sed and ExD hearts (P 0.001 and P = 0.003, respectively) compared with the Sham group, while the Ex group was partly protected. After IR, apoptosis was evident in Sed hearts (P = 0.016), while Ex and ExD hearts were protected. Data suggest that cardioprotective opioids are produced by the heart, but not by the soleus. After IR, the δ-opioid receptor may mediate, in part, cardioprotection against necrosis but not apoptosis.

What is the central question of this study? Does dietary quercetin enrichment improve biochemical and histological outcomes in hearts from mdx mice? What is the main finding and what is its importance? Biochemical and histological findings suggest that chronic quercetin feeding of mdx mice may improve mitochondrial function and attenuate tissue pathology. Patients with Duchenne muscular dystrophy suffer from cardiac pathology, which causes up to 40% of all deaths because of fibrosis and cardiac complications. Quercetin is a flavonol with anti-inflammatory and antioxidant effects and is also an activator of peroxisome proliferator-activated receptor γ coactivator 1α capable of antioxidant upregulation, mitochondrial biogenesis and prevention of cardiac complications. We sought to determine the extent to which dietary quercetin enrichment prevents (experiment 1) and rescues cardiac pathology (experiment 2) in mdx mice. In experiment 1, 3-week-old mdx mice were fed control chow (C3w6m, n = 10) or chow containing 0.2% quercetin for 6 months (Q3w6m, n = 10). In experiment 2, 3-month-old mdx mice were fed control chow (C3m6m, n = 10) or 0.2% chow containing 0.2% quercetin for 6 months (Q3m6m, n = 10). Hearts were excised for histological and biochemical analyses. In experiment 1, Western blot targets for mitochondrial biogenesis (cytochrome c, P = 0.007) and antioxidant expression (superoxide dismutase 2, P = 0.014) increased in Q3w6m mice compared with C3w6m. Histology revealed increased utrophin (P = 0.025) and decreased matrix metalloproteinase 9 abundance (P = 0.040) in Q3w6m mice compared with C3w6m. In experiment 2, relative (P = 0.023) and absolute heart weights (P = 0.020) decreased in Q3m6m mice compared with C3m6m. Indications of damage (Haematoxylin- and Eosin-stained sections, P = 0.007) and Western blot analysis of transforming growth factor β1 (P = 0.009) were decreased in Q3m6m mice. Six months of quercetin feeding increased a mitochondrial biomarker, antioxidant protein and utrophin and decreased matrix metalloproteinase 9 in young mice. Given that these adaptations are associated with attenuated cardiac pathology and damage, the present findings may indicate that dietary quercetin enrichment attenuates dystrophic cardiac pathology, but physiological confirmation is needed.

Cardiotoxicity associated with the use of doxorubicin (DOX), and other chemotherapeutics, limits their clinical potential. This study determined the pharmacokinetics and antitumor and cardioprotective activity of free and liposome encapsulated phenyl-2-aminoethyl-selenide (PAESe).The pharmacokinetics of free PAESe and PAESe encapsulated in liposomes (SSL-PAESe) were determined in rats using liquid chromatography tandem mass-spectrometry. The antitumor and cardioprotective effects were determined in a mouse xenograft model of human prostate (PC-3) cancer and cardiomyocytes (H9C2).The encapsulation of PAESe in liposomes increased the circulation half-life and area under the drug concentration time profile, and decreased total systemic clearance significantly compared to free PAESe. Free- and SSL-PAESe improved survival, decreased weight-loss and prevented cardiac hypertrophy significantly in tumor bearing and healthy mice following treatment with DOX at 5 and 12.5 mg/kg. In vitro studies revealed PAESe treatment altered formation of reactive oxygen species (ROS), cardiac hypertrophy and gene expression, i.e., atrial natriuretic peptide and myosin heavy chain complex beta, in H9C2 cells.Treatment with free and SSL-PAESe exhibited antitumor activity in a prostate xenograft model and mitigated DOX-mediated cardiotoxicity.

Diabetes and Alzheimer's disease share pathologic links toward cognitive deficits. Pharmacologic agonist of the nuclear receptor, peroxisomal proliferator-activating receptor gamma (PPARγ), that is, rosiglitazone (rosi), are insulin sensitizing agents that improve memory in Alzheimer's disease. However, direct molecular signaling targets that improve memory by PPARγ in the hippocampus have not been investigated. We compared outcomes from oral versus intracerebroventricular (ICV) administration of rosi on memory and changes in synaptic plasticity in type 2 diabetic (db/db) mice. Db/db mice treated with rosi (ICV) showed significant improvement in memory, long-term potentiation, and post-tetanic potentiation but did not improve peripheral insulin sensitivity. Gene and protein analysis revealed increased brain-derived neurotrophic factor (BDNF) in db/db mice treated with rosi (ICV). Transcriptional activation of exon IX as determined by luciferase assays confirmed PPARγ regulation of BDNF promoter activity. Transient transfection of constitutively active PPARγ plasmid in hippocampal neuronal cells induced increased BDNF, AMPA, and NMDA receptors expression and spine formation. Findings from the present study implicate a novel PPARγ-BDNF molecular signaling mechanism as a potential therapeutic target for cognitive impairment.

Insulin is secreted from the β-cells of the pancreas and helps maintain glucose homeostasis. Although secreted peripherally, insulin also plays a profound role in cognitive function. Increasing evidence suggests that insulin signaling in the brain is necessary to maintain health of neuronal cells, promote learning and memory, decrease oxidative stress, and ultimately increase neuronal survival. This chapter summarizes the different facets of insulin signaling necessary for learning and memory and additionally explores the association between cognitive impairment and central insulin resistance. The role of impaired insulin signaling in the advancement of cognitive dysfunction is relevant to the current debate of whether the shared pathophysiological mechanisms between diabetes and cognitive impairment implicate a direct relationship. Here, we summarize a vast amount of literature that suggests a strong association between impaired brain insulin signaling and cognitive impairment.

Patients with diabetes exhibit significantly altered renin-angiotensin system (RAS) control. Recently, it has been determined that hyperglycemic conditions induce an increase in angiotensin II (AT II) expression; specifically by cardiomyocytes. Altered RAS has been shown to be associated with an increase in oxidative stress and cardiac dysfunction leading to the development of cardiac hypertrophy. The transient outward potassium current (I(to)) in cardiac myocytes is mainly mediated by members of the Kv subfamily of voltage gated potassium channels and has been shown to be altered in cellular localization and expression during the development of cardiac hypertrophy. However it is not clear as to how AT II affects the pore forming complex at the cell membrane and thus directly affects the I(to) current. In the current study, we explored the protective effect of PPARγ ligands on cardiomyocyte I(to) by preventing NADPH Oxidase activation and the ensuing ROS formation. Furthermore, short term PPARγ activation in diabetic leptin deficient db/db mice displayed improvements in the membrane association of the molecular components of I(to) as well as prolonged QT interval. These findings demonstrate that PPARγ agonists have the potential to attenuate cardiomyocyte dysfunction associated with diabetes.

Accumulating evidence highlights intriguing interplays between circadian and metabolic pathways. We show that PER2 directly and specifically represses PPARγ, a nuclear receptor critical in adipogenesis, insulin sensitivity, and inflammatory response. PER2-deficient mice display altered lipid metabolism with drastic reduction of total triacylglycerol and nonesterified fatty acids. PER2 exerts its inhibitory function by blocking PPARγ recruitment to target promoters and thereby transcriptional activation. Whole-genome microarray profiling demonstrates that PER2 dictates the specificity of PPARγ transcriptional activity. Indeed, lack of PER2 results in enhanced adipocyte differentiation of cultured fibroblasts. PER2 targets S112 in PPARγ, a residue whose mutation has been associated with altered lipid metabolism. Lipidomic profiling demonstrates that PER2 is necessary for normal lipid metabolism in white adipocyte tissue. Our findings support a scenario in which PER2 controls the proadipogenic activity of PPARγ by operating as its natural modulator, thereby revealing potential avenues of pharmacological and therapeutic intervention. © 2010 Elsevier Inc.

In experimental animal and cell culture models, activation of peroxisome proliferator-activated receptor (PPAR) gamma in heart has been shown to have beneficial effects on cardiac function and cardiomyocyte physiology. The goal of this study was to identify the signaling pathway by which PPARgamma activation protects cardiomyocytes from the deleterious effects of hypertrophic stimuli. In primary cardiomyocyte cultures, we found that genetic or pharmacological activation of PPARgamma protected cells from cardiac hypertrophy induced by alpha-adrenergic stimulation. Examination of gene expression in these cells revealed a surprising increase in the expression of adiponectin in cardiomyocytes and secretion of the high-molecular-weight form of the hormone into media. Using RNAi to block PPARgamma-induced adiponectin production or adiponectin receptor gene expression, we found that the PPARgamma-mediated anti-hypertrophic effect required cardiomyocyte-produced adiponectin, as well as an intact adiponectin signaling pathway. Furthermore, mice expressing constitutive-active PPARgamma and cardiomyocyte specific adiponectin expression were protected from high-fat diet-induced cardiac hypertrophy and remodeling. These findings demonstrate that functional adiponectin hormone can be produced from the heart and raise the possibility that beneficial effects of PPARgamma activation in heart could be due in part to local production of adiponectin that acts on cardiomyocytes in an autocrine manner.

To better understand the role of insulin signaling in the development of Alzheimer's disease (AD), we utilized an animal model (intracerebroventricular injection of streptozotocin—ic-streptozotocin (STZ)) that displays insulin resistance only in the brain and exhibits AD pathology. In this model, deficits in hippocampal synaptic transmission and long-term potentiation (LTP) were observed. The decline in LTP correlated with decreased expression of NMDAR subunits NR2A and NR2B. The deficits in LTP were accompanied by changes in the expression and function of synaptic AMPARs. In ic-STZ animals, an alteration in integrin-linked kinase (ILK)-glycogen synthase kinase 3 beta (GSK-3-β) signaling was identified (p < 0.05). Similarly, there was decreased expression (p < 0.05) of brain derived neurotropic factor (BDNF) and stargazin, an AMPAR auxiliary subunit; both are required for driving AMPA receptors to the surface of the postsynaptic membrane. Our data illustrate that altered ILK-GSK-3β signaling due to impaired insulin signaling may decrease the trafficking and function of postsynaptic glutamate receptors; thereby, leading to synaptic deficits contributing to memory loss.

We recently reported that the activation of H-Ras represents one of the signaling steps underlying the interleukin-1beta (IL-1beta)-mediated metabolic dysfunction of the islet beta-cell. In the present study, we examined potential contributory roles of membrane-associated, cholesterol-enriched lipid rafts/caveolae and their constituent proteins (e.g., caveolin-1 [Cav-1]) as potential sites for IL-1beta-induced nitric oxide (NO) release in the isolated beta-cell. Disruption of lipid rafts (e.g., with cyclodextrin) markedly reduced IL-1beta-induced gene expression of inducible NO synthase (iNOS) and NO release from beta-cells. Immunologic and confocal microscopic evidence also suggested a transient but significant stimulation of tyrosine phosphorylation of Cav-1 in beta-cells briefly (for 15 min) exposed to IL-1beta that was markedly attenuated by three structurally distinct inhibitors of protein tyrosine phosphorylation. Overexpression of an inactive mutant of Cav-1 lacking the tyrosine phosphorylation site (Y14F) or an siRNA-mediated Cav-1 knock down also resulted in marked attenuation of IL-1beta-induced iNOS gene expression and NO release from these cells, thus further implicating Cav-1 in this signaling cascade. IL-1beta treatment also increased (within 20 min) the translocation of H-Ras into lipid rafts. Here we provide the first evidence to suggest that tyrosine phosphorylation of Cav-1 and subsequent interaction among members of the Ras signaling pathway within the membrane lipid microdomains represent early signaling mechanisms of IL-1beta in beta-cells.

Mastoparan, a tetradecapeptide from wasp venom, stimulates insulin secretion from the islet -cells, presumably via activation of trimeric G proteins. Herein, we used Clostridial toxins, which selectively modify and inactivate the Rho subfamily of G proteins, to examine whether mastoparan-induced insulin secretion also involves activation of these signaling proteins. Mastoparan, but not mastoparan 17 (an inactive analog of mastoparan), significantly stimulated insulin secretion from TC3 and INS-1 cells. Preincubation of TC3 cells with either Clostridium difficille toxin B, which inactivates Rho, Cdc42, and Rac, or Clostridium sordellii toxin, which inactivates Ras, Rap, and Rac, markedly attenuated the mastoparan-induced insulin secretion, implicating Rac in this phenomenon. Mastoparan-stimulated insulin secretion was resistant to GGTI-2147, a specific inhibitor of geranylgeranylation of Rho G proteins (e.g. Rac), suggesting that mastoparan induces direct activation of Rac via GTP/GDP exchange. This was confirmed by a pull-down assay that quantifies the binding of activated (i.e. GTP-bound) Rac to p21-activated kinase. However, glucose-induced insulin secretion from these cells was abolished by toxin B or GGTI-2147, suggesting that the geranylgeranylation step is critical for glucosestimulated secretion. Mastoparan significantly increased the translocation of cytosolic Rac and Cdc42 to the membrane fraction. Confocal light microscopy revealed a substantial degree of colocalization of Rac (and, to a lesser degree, Cdc42) with insulin in -cells exposed to mastoparan. Further, stable expression of a dominant negative (N17Rac) form of Rac into INS-1 cells resulted in a significant reduction in mastoparanstimulated insulin secretion from these cells. Taken together, our findings implicate Rho G proteins, specifically Rac, in mastoparan-induced insulin release. (Endocrinology 144: 4508 – 4518, 2003)

The majority of low molecular weight G proteins undergoes a series of post-translational modification steps, e.g., isoprenylation, at their C-terminal cysteine, which seem to be critical for the transport of the modified proteins to the membrane sites for interaction with their respective effector proteins. Using lovastatin, an inhibitor of mevalonic acid, and hence, isoprenoid biosynthesis, we demonstrated previously that protein isoprenylation is critical for physiological insulin secretion from normal rat islets. Herein, we used more selective synthetic inhibitors of protein prenylation to examine their effects on glucose- and calcium-mediated insulin secretion from betaTC3 cells. Both 3-allyl- and vinylfarnesols, which inhibit and/or modulate protein farnesyl transferases, significantly (80-95%) inhibited glucose- and KCl-stimulated insulin secretion from these cells. In a similar manner, the allyl and vinyl forms of geranylgeraniol, reagents targeted toward protein geranylation, attenuated insulin secretion elicited by glucose and KCl. Furthermore, manumycin A, a natural inhibitor of protein farnesylation, and geranylgeranyl transferase inhibitor-2147 (GGTI-2147), a peptidomimetic inhibitor of protein geranylgeranylation, also inhibited glucose- and KCl-induced insulin secretion to comparable degrees. Treatment of betaTC3 cells with either 3-vinylfarnesol or 3-vinyl geranylgeraniol resulted in accumulation of unprenylated proteins in the cytosolic fraction. These data further support our original formulation that inhibition of isoprenylation of small molecular weight G proteins might impede their interaction with their putative effectors, which may be required for physiological insulin secretion.

Objective: To study the effects of an aldose reductase inhibitor (ARI-509, Wyeth-Ayerst, Princeton, NJ) and aminoguanidine (AMG), agents that have been reported to prevent or delay diabetic retinopathy, on retinal vascular abnormalities and the immunocytochemical expression in the retina of vascular endothelial growth factor (VEGF) in rats maintained for up to 2 years on a 50% galactose diet. Methods: Albino rats were placed on a control diet, a diet containing 50% galactose, or the 50% galactose diet containing either ARI-509 or AMG. Treatment with ARI-509 or AMG was initiated at the beginning of the experiment or after 12 months of galactose feeding. After 22 to 24 months, the rats were killed and the retinal vasculature from half of one eye was isolated by trypsinelastase digestion for semiquantitative evaluation of retinal vascular lesions. The other half of the retina was prepared for immunocytochemistry and stained for the presence of VEGF, factor VIII, vimentin, and glial fibrillary acidic protein. Red blood cells, sciatic nerves, and a portion of the retina from the second eye were assayed for glucose, galactose, fructose, sorbitol, galactitol, and myo -inositol. Red blood cells were also assayed for galactosylated hemoglobin. Results: Galactose-fed animals developed a vascular retinopathy characterized by severe cellular loss in the retinal capillaries and intensification of periodic acid-Schiff staining of the vascular basement membranes. Some animals also displayed dilation and hypercellularity of vessels in the posterior retina. These changes were substantially reduced in animals receiving ARI-509 from the beginning of the galactose diet, but were unaffected in all of the other treatment groups. None of the rats receiving ARI-509 or AMG treatment, whether initiated from the onset or after 12 months of galactosemia, demonstrated VEGF immunoreactivity. With the exception of the animals receiving ARI-509 from the beginning of the experiment, all of the galactose-fed animals developed dense cataracts within 6 weeks of the beginning of the galactose diet. Galactitol levels in animals receiving ARI-509 were 86% to 93% lower in red blood cells, retina, and sciatic nerve than those in the other galactosefed groups. Conclusions: Although ARI-509 and AMG have different abilities to delay or prevent the diabetic-like retinopathy in galactosemic rats, even when substantial retinal microvascular acellularity occurs, both drugs prevent the immunocytochemical expression of VEGF. These results suggest that factors other than hypoxia may be responsible for VEGF expression in the retina, and that aldose reductase inhibitors and AMG have potential roles in preventing such expression and, thus, perhaps preventing retinal neovascularization.