Congenital adiponectin deficiency mitigates high-fat-diet-induced obesity in gonadally intact male and female, but not in ovariectomized mice.

Epidemiological literature indicates that women are less susceptible to type II diabetes (T2D) than males. The general consensus is that estrogen is protective, whereas its deficiency in post-menopause is associated with adiposity and impaired insulin sensitivity. However, epidemiological data suggests that males are more prone to developing T2D, and at a lower BMI, compared to females during post-menopausal years; suggesting that another factor, other than estrogen, protects females. We proposed to determine if adiponectin (APN) serves as this protective factor. An initial experiment was performed in which gonadally intact male and female mice were fed either a purified low-fat diet (LFD) or high-fat diet (HFD) (40% kcals from fat) for 16 weeks. An additional group of HFD ovariectomy (OVX) mice were included to assess estrogen deficiency's impact on obesity. Body composition, adipose tissue inflammation, ectopic lipid accumulation as well as glucose metabolism and insulin resistance were assessed. In corroboration with previous data, estrogen deficiency (OVX) exacerbated HFD-induced obesity in female mice. However, despite a higher body fat percentage and a similar degree of hepatic and skeletal muscle lipid accumulation, female OVX HFD-fed mice exhibited enhanced insulin sensitivity relative to HFD-fed males. Therefore, a subsequent HFD experiment was performed utilizing male and female (both gonadally intact and OVX) APN deficient mice (APN-/-) and wildtype littermates to determine if APN is the factor which protects OVX females from the similar degree of metabolic dysfunction as males in the setting of obesity. Indirect calorimetry was used to determine observed phenotype differences. APN deficiency limited adiposity and mitigated HFD-induced insulin resistance and adipose tissue inflammation in gonadally intact male and female, but not in OVX mice. Using indirect calorimetry, we uncovered that slight, but non-statistically significant differences in food intake and energy expenditure leading to a net difference in energy balance likely explain the reduced body weight exhibited by male APN-deficient mice. In conclusion, congenital APN deficiency is protective against obesity development in gonadally intact mice, however, in the setting of estrogen deficiency (OVX) this is not true. These findings suggest that gonadal status dictates the protective effects of congenital APN deficiency in the setting of HFD-induced obesity.

Characterization, bio-uptake and toxicity of polymer-coated silver nanoparticles and their interaction with human peripheral blood mononuclear cells.

Silver nanoparticles (AgNPs) are widely used in medical applications due to their antibacterial and antiviral properties. Despite the extensive study of AgNPs, their toxicity and their effect on human health is poorly understood, as a result of issues such as poor control of NP properties and lack of proper characterization. The aim of this study was to investigate the combined characterization, bio-uptake, and toxicity of well-characterized polyvinylpyrrolidone (PVP)-coated AgNPs in exposure media during exposure time using primary human cells (peripheral blood mononuclear cells (PBMCs)). AgNPs were synthesized in-house and characterized using a multimethod approach. Results indicated the transformation of NPs in RPMI medium with a change in size and polydispersity over 24 h of exposure due to dissolution and reprecipitation. No aggregation of NPs was observed in the RPMI medium over the exposure time (24 h). A dose-dependent relationship between PBMC uptake and Ag concentration was detected for both AgNP and AgNO3 treatment. There was approximately a two-fold increase in cellular Ag uptake in the AgNO3 vs the NP treatment. Cytotoxicity, using LDH and MTS assays and based on exposure concentrations was not significantly different when comparing NPs and Ag ions. Based on differential uptake, AgNPs were more toxic after normalizing toxicity to the amount of cellular Ag uptake. Our data highlights the importance of correct synthesis, characterization, and study of transformations to obtain a better understanding of NP uptake and toxicity. Statistical analysis indicated that there might be an individual variability in response to NPs, although more research is required.

Neurofascin Is a Novel Component of Rod Photoreceptor Synapses in the Outer Retina.

Neural circuit formation is an intricate and complex process where multiple neuron types must come together to form synaptic connections at a precise location and time. How this process is orchestrated during development remains poorly understood. Cell adhesion molecules are known to play a pivotal role in assembling neural circuits. They serve as recognition molecules between corresponding synaptic partners. In this study, we identified a new player in assembling neural circuits in the outer retina, the L1-family cell adhesion molecule Neurofascin (Nfasc). Our data reveals Nfasc is expressed in the synaptic layer where photoreceptors make synaptic connections to their respective partners. A closer examination of Nfasc expression shows high levels of expression in rod bipolars but not in cone bipolars. Disruption of Nfasc using a conditional knockout allele results in selective loss of pre- and post-synaptic proteins in the rod synaptic layer but not in the cone synaptic layer. Electron microscopic analysis confirms that indeed there are abnormal synaptic structures with less dendrites of rod bipolars innervating rod terminals in loss of Nfasc animals. Consistent with these findings, we also observe a decrease in rod-driven retinal responses with disruption of Nfasc function but not in cone-driven responses. Taken together, our data suggest a new role of Nfasc in rod synapses within the mouse outer retina.

Three-layered silver nanoparticles to trace dissolution and association to a green alga.

Core-shell silver nanoparticles (NPs) consisting of an inner Ag core and successive layers of Au and Ag (Ag@Au@Ag) were used to measure the simultaneous association of Ag NPs and ionic Ag by the green alga Chlamydomonas (C.) reinhardtii. Dissolution of the inner Ag core was prevented by a gold (Au) layer, while the outer Ag layer was free to dissolve. In short-term experiments, we exposed C. reinhardtii to a range of environmentally realistic Ag concentrations added as AgNO3 or as NPs. Results provide three lines of evidence for the greater cell-association of NPs compared to dissolved Ag over the concentration range tested, assuming that cell-association comprises both uptake and adsorption. First, the cell-association rate constants (kuw) for total Ag (AgNP+D), NPs (AgNP) and AuNP were similar and 2.2-fold higher than the one from AgD exposure, suggesting predominant association of the particles over the dissolved form. Second, model calculations based on Ag fluxes suggested that only 6-33% of algal burden was from AgD. Third, the significantly lower AgNP/Au ratio measured with the algae after exposure (2.1 ± 0.1) compared to the AgNP/Au ratio of the NPs in the media (2.47 ± 0.05) suggests cell-association of NPs depleted in Ag. Core-shell NPs provide an innovative tool to understand NP behavior and to directly delineate Ag accumulation from ion and NPs in aquatic systems.

NADPH Oxidase-Derived Peroxynitrite Drives Inflammation in Mice and Human Nonalcoholic Steatohepatitis via TLR4-Lipid Raft Recruitment.

The molecular events that link NADPH oxidase activation and the induction of Toll-like receptor (TLR)-4 recruitment into hepatic lipid rafts in nonalcoholic steatohepatitis (NASH) are unclear. We hypothesized that in liver, NADPH oxidase activation is key in TLR4 recruitment into lipid rafts, which in turn up-regulates NF-κB translocation to the nucleus and subsequent DNA binding, leading to NASH progression. Results from confocal microscopy showed that liver from murine and human NASH had NADPH oxidase activation, which led to the formation of highly reactive peroxynitrite, as shown by 3-nitrotyrosine formation in diseased liver. Expression and recruitment of TLR4 into the lipid rafts were significantly greater in rodent and human NASH. The described phenomenon was NADPH oxidase, p47phox, and peroxynitrite dependent, as liver from p47phox-deficient mice and from mice treated with a peroxynitrite decomposition catalyst [iron(III) tetrakis(p-sulfonatophenyl)porphyrin] or a peroxynitrite scavenger (phenylboronic acid) had markedly less Tlr4 recruitment into lipid rafts. Mechanistically, peroxynitrite-induced TLR4 recruitment was linked to increased IL-1ß, sinusoidal injury, and Kupffer cell activation while blocking peroxynitrite-attenuated NASH symptoms. The results strongly suggest that NADPH oxidase-mediated peroxynitrite drove TLR4 recruitment into hepatic lipid rafts and inflammation, whereas the in vivo use of the peroxynitrite scavenger phenylboronic acid, a novel synthetic molecule having high reactivity with peroxynitrite, attenuates inflammatory pathogenesis in NASH.

Upregulation of miR21 and repression of Grhl3 by leptin mediates sinusoidal endothelial injury in experimental nonalcoholic steatohepatitis.

Sinusoidal endothelial dysfunction (SED) has been found to be an early event in nonalcoholic steatohepatitis (NASH) progression but the molecular mechanisms underlying its causation remains elusive. We hypothesized that adipokine leptin worsens sinusoidal injury by decreasing functionally active nitric oxide synthase 3 (NOS)3 via miR21. Using rodent models of NASH, and transgenic mice lacking leptin and leptin receptor, results showed that hyperleptinemia caused a 4-5 fold upregulation of hepatic miR21 as assessed by qRTPCR. The upregulation of miR21 led to a time-dependent repression of its target protein Grhl3 levels as shown by western blot analyses. NOS3-p/NOS3 ratio which is controlled by Grhl3 was significantly decreased in NASH models. SED markers ICAM-1, VEGFR-2, and E-selectin as assessed by immunofluorescence microscopy were significantly up regulated in the progressive phases of NASH. Lack of leptin or its receptor in vivo, reversed the upregulation of miR21 and restored the levels of Grhl3 and NOS3-p/NOS3 ratio coupled with decreased SED dysfunction markers. Interestingly, leptin supplementation in mice lacking leptin, significantly enhanced miR21 levels, decreased Grhl3 repression and NOS3 phosphorylation. Leptin supplementation in isolated primary endothelial cells, Kupffer cells and stellate cells showed increased mir21 expression in stellate cells while sinusoidal injury was significantly higher in all cell types. Finally miR21 KO mice showed increased NOS3-p/NOS3 ratio and reversed SED markers in the rodent models of NASH. The experimental results described here show a close association of leptin-induced miR21 in aiding sinusoidal injury in NASH.

M1 polarization bias and subsequent nonalcoholic steatohepatitis progression is attenuated by nitric oxide donor DETA NONOate via inhibition of CYP2E1-induced oxidative stress in obese mice.

Activation of M1 macrophages in nonalcoholic steatohepatitis (NASH) is produced by several external or endogenous factors: inflammatory stimuli, oxidative stress, and cytokines are known. However, any direct role of oxidative stress in causing M1 polarization in NASH has been unclear. We hypothesized that CYP2E1-mediated oxidative stress causes M1 polarization in experimental NASH, and that nitric oxide (NO) donor administration inhibits CYP2E1-mediated inflammation with concomitant attenuation of M1 polarization. Because CYP2E1 takes center stage in these studies, we used a toxin model of NASH that uses a ligand and a substrate of CYP2E1 for inducing NASH. Subsequently, we used a methionine and choline-deficient diet-induced rodent NASH model where the role of CYP2E1 in disease progression has been shown. Our results show that CYP2E1 causes M1 polarization bias, which includes a significant increase in interleukin-1ß (IL-1ß) and IL-12 in both models of NASH, whereas CYP2E1-null mice or diallyl sulfide administration prevented it. Administration of gadolinium chloride (GdCl3), a macrophage toxin, attenuated both the initial M1 response and the subsequent M2 response, showing that the observed increase in cytokine levels is primarily from macrophages. Based on the evidence of an adaptive NO increase, the NO donor administration in vivo that mechanistically inhibited CYP2E1 catalyzed the oxidative stress during the entire study in NASH-abrogated M1 polarization and NASH progression. The results obtained show the association of CYP2E1 in M1 polarization, and that inhibition of CYP2E1 catalyzed oxidative stress by an NO donor (DETA NONOate [(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate]) can be a promising therapeutic strategy in NASH.

Micro-RNA 21 inhibition of SMAD7 enhances fibrogenesis via leptin-mediated NADPH oxidase in experimental and human nonalcoholic steatohepatitis.

Hepatic fibrosis in nonalcoholic steatohepatitis (NASH) is the common pathophysiological process resulting from chronic liver inflammation and oxidative stress. Although significant research has been carried out on the role of leptin-induced NADPH oxidase in fibrogenesis, the molecular mechanisms that connect the leptin-NADPH oxidase axis in upregulation of transforming growth factor (TGF)-ß signaling have been unclear. We aimed to investigate the role of leptin-mediated upregulation of NADPH oxidase and its subsequent induction of micro-RNA 21 (miR21) in fibrogenesis. Human NASH livers and a high-fat (60% kcal) diet-fed chronic mouse model, where hepatotoxin bromodichloromethane was used to induce NASH, were used for this study. To prove the role of the leptin-NADPH oxidase-miR21 axis, mice deficient in genes for leptin, p47phox, and miR21 were used. Results showed that wild-type mice and human livers with NASH had increased oxidative stress, increased p47phox expression, augmented NF-κB activation, and increased miR21 levels. These mice and human livers showed increased TGF-ß, SMAD2/3-SMAD4 colocalizations in the nucleus, increased immunoreactivity against Col1α, and α-SMA with a concomitant decrease in protein levels of SMAD7. Mice that were deficient in leptin or p47phox had decreased activated NF-κB and miR21 levels, suggesting the role of leptin and NADPH oxidase in inducing NF-κB-mediated miR21 expression. Further miR21 knockout mice had decreased colocalization events of SMAD2/3-SMAD4 in the nucleus, increased SMAD7 levels, and decreased fibrogenesis. Taken together, the studies show the novel role of leptin-NADPH oxidase induction of miR21 as a key regulator of TGF-ß signaling and fibrogenesis in experimental and human NASH.