Pharmacological Inhibition of NOX4 Improves Mitochondrial Function and Survival in Human Beta-Cells

Previous studies have reported beneficial effects of NADPH oxidase 4 (NOX4) inhibition on beta-cell survival in vitro and in vivo. The mechanisms by which NOX4 inhibition protects insulin producing cells are, however, not known. The aim of the present study was to investigate the effects of a pharmacological NOX4 inhibitor (GLX7013114) on human islet and EndoC-βH1 cell mitochondrial function, and to correlate such effects with survival in islets of different size, activity, and glucose-stimulated insulin release responsiveness. We found that maximal oxygen consumption rates, but not the rates of acidification and proton leak, were increased in islets after acute NOX4 inhibition. In EndoC-βH1 cells, NOX4 inhibition increased the mitochondrial membrane potential, as estimated by JC-1 fluorescence; mitochondrial reactive oxygen species (ROS) production, as estimated by MitoSOX fluorescence; and the ATP/ADP ratio, as assessed by a bioluminescent assay. Moreover, the insulin release from EndoC-βH1 cells at a high glucose concentration increased with NOX4 inhibition. These findings were paralleled by NOX4 inhibition-induced protection against human islet cell death when challenged with high glucose and sodium palmitate. The NOX4 inhibitor protected equally well islets of different size, activity, and glucose responsiveness. We conclude that pharmacological alleviation of NOX4-induced inhibition of beta-cell mitochondria leads to increased, and not decreased, mitochondrial ROS, and this was associated with protection against cell death occurring in different types of heterogeneous islets. Thus, NOX4 inhibition or modulation may be a therapeutic strategy in type 2 diabetes that targets all types of islets.

Morroniside Promotes PGC-1α-Mediated Cholesterol Efflux in Sodium Palmitate or High Glucose-Induced Mouse Renal Tubular Epithelial Cells

Lipid deposition is an etiology of renal damage caused by lipid metabolism disorder in diabetic nephropathy (DN). Thus, reducing lipid deposition is a feasible strategy for the treatment of DN. Morroniside (MOR), an iridoid glycoside isolated from the Chinese herb Cornus officinalis Sieb. et Zucc., is considered to be an effective drug in inhibiting oxidative stress, reducing inflammatory response, and countering apoptosis. To explore the protective mechanism of MOR in attenuating renal lipotoxicity in DN, we investigated the effect of MOR on an in vitro model of lipid metabolism disorder of DN established by stimulating mouse renal tubular epithelial cells (mRTECs) with sodium palmitate (PA) or high glucose (HG). Oil Red O and filipin cholesterol staining assays were used to determine intracellular lipid accumulation status. Results revealed that PA or HG stimulation inhibited the expressions of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), liver X receptors (LXR), ATP-binding cassette subfamily A member 1 (ABCA1), ABCG1, and apolipoprotein E (ApoE) in mRTECs as evidenced by western blot and quantitative real-time PCR, resulting in increased intracellular lipid deposition. Interestingly, MOR upregulated expressions of PGC-1α, LXR, ABCA1, ABCG1, and ApoE, thus reducing cholesterol accumulation in mRTECs, suggesting that MOR might promote cholesterol efflux from mRTECs via the PGC-1α/LXR pathway. Of note, silencing PGC-1α reversed the promotive effect of MOR on PA- or HG-induced cellular cholesterol accumulation. In conclusion, our results suggest that MOR has a protective effect on mRTECs under high lipid or high glucose conditions, which may be related to the promotion of intracellular cholesterol efflux mediated by PGC-1α.

Abstract P738: Matrix Metalloproteinase-3 (mmp3) Activation Impairs Endothelial Integrity and Increases Vascular Injury After Ischemic Stroke in Female Diabetic Rats

Introduction: Females patients with diabetes suffer from poor outcomes of ischemic stroke but underlying reasons are not fully understood. We have shown that 1) adult female diabetic rats develop greater infarct and hemorrhagic transformation (HT) leading to poorer outcomes after ischemic stroke, and 2) MMP3 activity is increased to a greater extent in female endothelial cells as well as the microvasculature of diabetic female rats than in males. This led us to hypothesize that MMP3 mediates disruption of endothelial integrity amplifying vascular injury in female diabetic rats. Methods: Diabetic female Wistar rats, subjected to 60 min MCAO, received a single dose of MMP3 inhibitor (UK356618; 15mg/kg; iv) or vehicle after reperfusion. On Day 3, adhesive removal time (ART), behavioral composite score, brain infarct size, edema and macroscopic HT were recorded. Primary brain microvascular endothelial cells (BMVECs) isolated from female rats were cultured in 25mM glucose plus 100μM sodium palmitate for 48 hours followed by hypoxia insult for 24 hours in the presence/absence of UK356618 (20nM). Endothelial phenotype and integrity were assessed. Results: Brain edema and HT scores were lower and outcomes were improved with MMP3 inhibition but infarct size was not different between the groups (Table). Hypoxia decreased tight junction protein occludin-1 while increasing transforming growth factor receptor-1 (TGF-R1), a key modulator of endothelial phenotype. Treatment with inhibitor UK356618 reversed these responses. Conclusions: The lack of a difference in infarct size with the treatment suggests that MMP3 inhibition improves short term outcomes in female diabetic rats via preservation of endothelial integrity. The decrease in TGF-R1 needs to be further investigated for long term effects of MMP3 inhibition on vascular restoration and recovery after stroke in diabetes.

Insecticidal Activity of Commiphora erythraea Essential Oil and Its Emulsions Against Larvae of Three Mosquito Species

The use of essential oils as ecofriendly tools for vector management is one of the mainstreams for biopesticide research. We evaluated the larvicidal properties of Commiphora erythraea (opoponax) essential oil and its fractions against Culex restuans Theobald, Culex pipiens L., and Aedes aegypti L. The use of bio-based amylose–N-1-hexadecylammonium chloride inclusion complex (Hex-Am) and amylose–sodium palmitate inclusion complex (Na-Palm) as emulsifiers for C. erythraea essential oil was also investigated. Bisabolene was the most abundant chemical constituent in the whole essential oil (33.9%), fraction 2 (62.5%), and fraction 4 (23.8%) while curzerene (32.6%) and α-santalene (30.1%) were the dominant chemical constituents in fractions 1 and 3, respectively. LC50 values for the whole essential oil were 19.05 ppm for Cx. restuans, 22.61 ppm for Cx. pipiens, and 29.83 ppm for Ae. aegypti and differed significantly. None of the four C. erythraea essential oil fractions were active against mosquito larvae. Two CYP450 genes (CYP6M11 and CYP6N12) and one GST gene (GST-2) were significantly upregulated in Ae. aegypti larvae exposed to C. erythraea essential oil suggesting their potential involvement in metabolic pathways for C. erythraea essential oil. Essential oil emulsions produced with Hex-Am were more toxic than the whole essential oil while those produced with Na-Palm had similar toxicity as the whole essential oil. These findings demonstrate that C. erythraea essential oil is a promising source of mosquito larvicide and that the use of Hex-Am as an emulsifier can enhance the insecticidal properties of C. erythraea essential oil.

tBHQ Induces a Hormetic Response That Protects L6 Myoblasts against the Toxic Effect of Palmitate

Nutritional status, in particular overweight and obesity, as well as sedentarism and high-fat diet consumption, are important risk factors to develop chronic diseases, which have a higher impact on the elderly’s health. Therefore, these nutritional problems have become a concern to human healthspan and longevity. The fatty acids obtained thru the diet or due to fatty acid synthesis during obesity accumulate within the body generating toxicity and cell death. Fat is not only stored in adipose tissue, but it can also be stored in skeletal muscle. Palmitic acid (PA) has been reported as one of the most important saturated free fatty acids; it is associated to chronic oxidative stress and increased mitochondrial ROS production causing cell death by apoptosis. In skeletal muscle, palmitate has been associated with various pathophysiological consequences, which lead to muscle deterioration during aging and obesity. Since molecules that modify redox state have been proven to prevent cellular damage by inducing a hormetic response, the aim of this study was to evaluate if tert-butylhydroquinone (tBHQ) could activate an antioxidant hormetic response that would be able to protect L6 myoblasts from palmitate toxic effect. Our results provide evidence that tBHQ is able to protect L6 myoblasts against the toxicity induced by sodium palmitate due to a synergistic activation of different signaling pathways such as Nrf2 and NF-κB.

Amylose Inclusion Complexes as Emulsifiers for Garlic and Asafoetida Essential Oils for Mosquito Control

Although the insecticidal properties of some plant essential oils are well-documented, their use in integrated pest and vector management is complicated by their high volatility, low thermal stability, high sensitivity to oxidation, and low solubility in water. We investigated the use of bio-based N-1-hexadecylammonium chloride and sodium palmitate amylose inclusion complexes as emulsifiers for two essential oils, garlic and asafoetida, known to be highly toxic to mosquito larvae. Four emulsions of each essential oil based on amylose hexadecylammonium chloride and amylose sodium palmitate inclusion complexes were evaluated for their toxicity against Aedes aegypti L. larvae relative to bulk essential oils. All emulsions were significantly more toxic than the bulk essential oil with the lethal dosage ratios ranging from 1.09–1.30 relative to bulk essential oil. Droplet numbers ranged from 1.11 × 109 to 9.55 × 109 per mL and did not change significantly after a 6-month storage period. These findings demonstrated that amylose inclusion complexes enhanced the toxicity of essential oils and could be used to develop new essential oil based larvicides for use in integrated vector management.