Adipose tissue macrophages (ATMs) are involved in the development of insulin resistance in obesity. We have recently shown that myeloid cell–specific reduction of HMG-CoA reductase (Hmgcr^m–/m–), which is the rate-limiting enzyme in cholesterol biosynthesis, protects against atherosclerosis by inhibiting macrophage migration in mice. We hypothesized that ATMs are harder to accumulate in Hmgcr^m–/m– mice than in control Hmgcr^fl/fl mice in the setting of obesity. To test this hypothesis, we fed Hmgcr^m–/m– and Hmgcr^fl/fl mice a high-fat diet (HFD) for 24 weeks and compared plasma glucose metabolism as well as insulin signaling and histology between the two groups. Myeloid cell–specific reduction of Hmgcr improved glucose tolerance and insulin sensitivity without altering body weight in the HFD-induced obese mice. The improvement was due to a decrease in the number of ATMs. The ATMs were reduced by decreased recruitment of macrophages as a result of their impaired chemotactic activity. These changes were associated with decreased expression of proinflammatory cytokines in adipose tissues. Myeloid cell–specific reduction of Hmgcr also attenuated hepatic steatosis. In conclusion, reducing myeloid HMGCR may be a promising strategy to improve insulin resistance and hepatic steatosis in obesity.

Richard S Surwit
Sep 1, 1988; 37:1163-1167
Original Article

Patrice D. Cani
Jun 1, 2008; 57:1470-1481
Metabolism

Branched esters of palmitic acid and hydroxystearic acid (PAHSA) are anti-inflammatory and antidiabetic lipokines that connect glucose and lipid metabolism. We aimed to characterize involvement of the 5-PAHSA regioisomer in the adaptive metabolic response of white adipose tissue (WAT) to cold exposure (CE) in mice, exploring the cross talk between glucose utilization and lipid metabolism. CE promoted local production of 5- and 9-PAHSAs in WAT. Metabolic labeling of de novo lipogenesis (DNL) using ²H₂O revealed that 5-PAHSA potentiated the effects of CE and stimulated triacylglycerol (TAG)/fatty acid (FA) cycling in WAT through impacting lipogenesis and lipolysis. Adipocyte lipolytic products were altered by 5-PAHSA through selective FA re-esterification. The impaired lipolysis in global adipose triglyceride lipase (ATGL) knockout mice reduced free PAHSA levels and uncovered a metabolite reservoir of TAG-bound PAHSAs (TAG estolides) in WAT. Utilization of ¹³C isotope tracers and dynamic metabolomics documented that 5-PAHSA primes adipocytes for glucose metabolism in a different way from insulin, promoting DNL and impeding TAG synthesis. In summary, our data reveal new cellular and physiological mechanisms underlying the beneficial effects of 5-PAHSA and its relation to insulin action in adipocytes and independently confirm a PAHSA metabolite reservoir linked to ATGL-mediated lipolysis.

Schwann cell–derived exosomes communicate with dorsal root ganglia (DRG) neurons. The current study investigated the therapeutic effect of exosomes derived from healthy Schwann cells (SC-Exos) on diabetic peripheral neuropathy (DPN). We found that intravenous administration of SC-Exos to type 2 diabetic db/db mice with peripheral neuropathy remarkably ameliorated DPN by improving sciatic nerve conduction velocity and increasing thermal and mechanical sensitivity. These functional improvements were associated with the augmentation of epidermal nerve fibers and remyelination of sciatic nerves. Quantitative RT-PCR and Western blot analysis of sciatic nerve tissues showed that SC-Exo treatment reversed diabetes-reduced mature form of miRNA (miR)-21, -27a, and -146a and diabetes-increased semaphorin 6A (SEMA6A); Ras homolog gene family, member A (RhoA); phosphatase and tensin homolog (PTEN); and nuclear factor-B (NF-B). In vitro data showed that SC-Exos promoted neurite outgrowth of diabetic DRG neurons and migration of Schwann cells challenged by high glucose. Collectively, these novel data provide evidence that SC-Exos have a therapeutic effect on DPN in mice and suggest that SC-Exo modulation of miRs contributes to this therapy.

Diabetes triggers peripheral nerve alterations at a structural and functional level, collectively referred to as diabetic peripheral neuropathy (DPN). This work highlights the role of the liver X receptor (LXR) signaling pathway and the cross talk with the reactive oxygen species (ROS)–producing enzyme NADPH oxidase-4 (Nox4) in the pathogenesis of DPN. Using type 1 diabetic (T1DM) mouse models together with cultured Schwann cells (SCs) and skin biopsies from patients with type 2 diabetes (T2DM), we revealed the implication of LXR and Nox4 in the pathophysiology of DPN. T1DM animals exhibit neurophysiological defects and sensorimotor abnormalities paralleled by defective peripheral myelin gene expression. These alterations were concomitant with a significant reduction in LXR expression and increase in Nox4 expression and activity in SCs and peripheral nerves, which were further verified in skin biopsies of patients with T2DM. Moreover, targeted activation of LXR or specific inhibition of Nox4 in vivo and in vitro to attenuate diabetes-induced ROS production in SCs and peripheral nerves reverses functional alteration of the peripheral nerves and restores the homeostatic profiles of MPZ and PMP22. Taken together, our findings are the first to identify novel, key mediators in the pathogenesis of DPN and suggest that targeting LXR/Nox4 axis is a promising therapeutic approach.

Central to the development of diabetic macro- and microvascular disease is endothelial dysfunction, which appears well before any clinical sign but, importantly, is potentially reversible. We previously demonstrated that hyperglycemia activates nuclear factor of activated T cells (NFAT) in conduit and medium-sized resistance arteries and that NFAT blockade abolishes diabetes-driven aggravation of atherosclerosis. In this study, we test whether NFAT plays a role in the development of endothelial dysfunction in diabetes. NFAT-dependent transcriptional activity was elevated in skin microvessels of diabetic Akita (Ins2^+/–) mice when compared with nondiabetic littermates. Treatment of diabetic mice with the NFAT blocker A-285222 reduced NFATc3 nuclear accumulation and NFAT-luciferase transcriptional activity in skin microvessels, resulting in improved microvascular function, as assessed by laser Doppler imaging and iontophoresis of acetylcholine and localized heating. This improvement was abolished by pretreatment with the nitric oxide (NO) synthase inhibitor l-N^G-nitro-l-arginine methyl ester, while iontophoresis of the NO donor sodium nitroprusside eliminated the observed differences. A-285222 treatment enhanced dermis endothelial NO synthase expression and plasma NO levels of diabetic mice. It also prevented induction of inflammatory cytokines interleukin-6 and osteopontin, lowered plasma endothelin-1 and blood pressure, and improved mouse survival without affecting blood glucose. In vivo inhibition of NFAT may represent a novel therapeutic modality to preserve endothelial function in diabetes.

Masaki Yamamoto
May 22, 2019; 39:4179-4192
Neurobiology of Disease

Peter Klivenyi
Jan 1, 2000; 20:1-7
Cellular

Lennart Mucke
Jun 1, 2000; 20:4050-4058
Cellular

Giving birth triggers a wide repertoire of physiological and behavioral changes in the mother to enable her to feed and care for her offspring. These changes require coordination and are often orchestrated from the CNS, through as of yet poorly understood mechanisms. A neuronal population with a central role in puerperal changes is the tuberoinfundibular dopamine (TIDA) neurons that control release of the pituitary hormone, prolactin, which triggers key maternal adaptations, including lactation and maternal care. Here, we used Ca²⁺ imaging on mice from both sexes and whole-cell recordings on female mouse TIDA neurons in vitro to examine whether they adapt their cellular and network activity according to reproductive state. In the high-prolactin state of lactation, TIDA neurons shift to faster membrane potential oscillations, a reconfiguration that reverses upon weaning. During the estrous cycle, however, which includes a brief, but pronounced, prolactin peak, oscillation frequency remains stable. An increase in the hyperpolarization-activated mixed cation current, I_h, possibly through unmasking as dopamine release drops during nursing, may partially explain the reconfiguration of TIDA rhythms. These findings identify a reversible plasticity in hypothalamic network activity that can serve to adapt the dam for motherhood.

SIGNIFICANCE STATEMENT Motherhood requires profound behavioral and physiological adaptations to enable caring for offspring, but the underlying CNS changes are poorly understood. Here, we show that, during lactation, neuroendocrine dopamine neurons, the "TIDA" cells that control prolactin secretion, reorganize their trademark oscillations to discharge in faster frequencies. Unlike previous studies, which typically have focused on structural and transcriptional changes during pregnancy and lactation, we demonstrate a functional switch in activity and one that, distinct from previously described puerperal modifications, reverses fully on weaning. We further provide evidence that a specific conductance (I_h) contributes to the altered network rhythm. These findings identify a new facet of maternal brain plasticity at the level of membrane properties and consequent ensemble activity.

To determine whether Cav1.2 voltage-gated Ca²⁺ channels contribute to astrocyte activation, we generated an inducible conditional knock-out mouse in which the Cav1.2 α subunit was deleted in GFAP-positive astrocytes. This astrocytic Cav1.2 knock-out mouse was tested in the cuprizone model of myelin injury and repair which causes astrocyte and microglia activation in the absence of a lymphocytic response. Deletion of Cav1.2 channels in GFAP-positive astrocytes during cuprizone-induced demyelination leads to a significant reduction in the degree of astrocyte and microglia activation and proliferation in mice of either sex. Concomitantly, the production of proinflammatory factors such as TNFα, IL1β and TGFβ1 was significantly decreased in the corpus callosum and cortex of Cav1.2 knock-out mice through demyelination. Furthermore, this mild inflammatory environment promotes oligodendrocyte progenitor cells maturation and myelin regeneration across the remyelination phase of the cuprizone model. Similar results were found in animals treated with nimodipine, a Cav1.2 Ca²⁺ channel inhibitor with high affinity to the CNS. Mice of either sex injected with nimodipine during the demyelination stage of the cuprizone treatment displayed a reduced number of reactive astrocytes and showed a faster and more efficient brain remyelination. Together, these results indicate that Cav1.2 Ca²⁺ channels play a crucial role in the induction and proliferation of reactive astrocytes during demyelination; and that attenuation of astrocytic voltage-gated Ca²⁺ influx may be an effective therapy to reduce brain inflammation and promote myelin recovery in demyelinating diseases.

SIGNIFICANCE STATEMENT Reducing voltage-gated Ca²⁺ influx in astrocytes during brain demyelination significantly attenuates brain inflammation and astrocyte reactivity. Furthermore, these changes promote myelin restoration and oligodendrocyte maturation throughout remyelination.

Mice, rats, or hamsters are immunized by giving biweekly injections of a purified antigen, cultured cells, or cDNA. For mice, if a pure, soluble protein antigen is being used and is abundant, a dose of 50–100 µg in adjuvant at each immunization is a sensible general recommendation; for rats and hamsters, a dose of 100–200 µg is sufficient. Lower doses can be used for antigens with higher immunogenicity. Adjuvants (Freund's, Ribi, Hunter's TiterMax, ImmunEasy, or Alum) should be mixed with the immunizing antigen for the first two immunizations only; Complete Freund's adjuvant is only used with the first immunization. Subsequent immunizations are performed in phosphate-buffered saline (PBS) or normal saline, with or without Incomplete Freund's adjuvant. The choice of adjuvant is dependent on the subclass of immunoglobulin required. Over the course of the 6-wk immunization schedule, each animal usually receives a total of six injections (three subcutaneous and three intraperitoneal). Once a good titer has developed against the antigen of interest, regular boosts and bleeds are performed to collect the maximum amount of serum. For rats and hamsters, boosts should be spaced every 2–3 wk, and serum samples of 400–500 µL should be collected 10–12 d after each boost. For mice, boosts should be spaced every 2–3 wk, and serum samples of 200–300 µL should be collected 10–12 d after each boost.

We visit the grassroots club where it all began for the EURO '96 finalist and UEFA Champions League winner.

Mycobacterium abscessus lung infections remain difficult to treat. Recent studies have recognized the power of new combinations of antibiotics such as bedaquiline and imipenem although in vitro data have questioned this combination. We report that the efficacy of the bedaquiline plus imipenem treatment relies essentially on the activity of bedaquiline in a C3HeB/FeJ mice model of infection with a rough variant of M. abscessus. The addition of imipenem contributed at clearing the infection in the spleen.

Title: Researchers Rejuvenate Blood-Forming Stem Cells in Mice
Category: Health News
Created: 5/3/2012 2:05:00 PM
Last Editorial Review: 5/4/2012 12:00:00 AM

Title: Lab Mice Stressed Out By Men, But Not Women, Study Finds
Category: Health News
Created: 4/28/2014 2:35:00 PM
Last Editorial Review: 4/29/2014 12:00:00 AM

Title: Scientists Pinpoint Brain's 'Hunger Circuit' in Mice
Category: Health News
Created: 4/28/2015 12:00:00 AM
Last Editorial Review: 4/28/2015 12:00:00 AM

Title: Transplanted Skin Stem Cells Help Blind Mice See Light
Category: Health News
Created: 4/21/2020 12:00:00 AM
Last Editorial Review: 4/22/2020 12:00:00 AM

Many clinical studies and epidemiological investigations have clearly demonstrated that women are twice as likely to develop cholesterol gallstones as men, and oral contraceptives and other estrogen therapies dramatically increase that risk. Further, animal studies have revealed that estrogen promotes cholesterol gallstone formation through the estrogen receptor (ER) α, but not ERβ, pathway. More importantly, some genetic and pathophysiological studies have found that G protein-coupled estrogen receptor (GPER) 1 is a new gallstone gene, Lith18, on chromosome 5 in mice and produces additional lithogenic actions, working independently of ERα, to markedly increase cholelithogenesis in female mice. Based on computational modeling of GPER, a novel series of GPER-selective antagonists were designed, synthesized, and subsequently assessed for their therapeutic effects via calcium mobilization, cAMP, and ERα and ERβ fluorescence polarization binding assays. From this series of compounds, one new compound, 2-cyclohexyl-4-isopropyl-N-(4-methoxybenzyl)aniline (CIMBA), exhibits superior antagonism and selectivity exclusively for GPER. Furthermore, CIMBA reduces the formation of 17β-estradiol-induced gallstones in a dose-dependent manner in ovariectomized mice fed a lithogenic diet for 8 weeks. At 32 μg/day/kg CIMBA, no gallstones are found, even in ovariectomized ERα (^–/–) mice treated with 6 μg/day 17β-estradiol and fed the lithogenic diet for 8 weeks. In conclusion, CIMBA treatment protects against the formation of estrogen-induced cholesterol gallstones by inhibiting the GPER signaling pathway in female mice. CIMBA may thus be a new agent for effectively treating cholesterol gallstone disease in women.­

Fatty liver involves ectopic lipid accumulation and dysregulated hepatic oxidative metabolism, which can progress to a state of elevated inflammation and fibrosis referred to as nonalcoholic steatohepatitis (NASH). The factors that control progression from simple steatosis to NASH are not fully known. Here, we tested the hypothesis that dietary vitamin E (VitE) supplementation would prevent NASH progression and associated metabolic alterations induced by a Western diet (WD). Hyperphagic melanocortin-4 receptor-deficient (MC4R^–/–) mice were fed chow, chow+VitE, WD, or WD+VitE starting at 8 or 20 weeks of age. All groups exhibited extensive hepatic steatosis by the end of the study (28 weeks of age). WD feeding exacerbated liver disease severity without inducing proportional changes in liver triglycerides. Eight weeks of WD accelerated liver pyruvate cycling, and 20 weeks of WD extensively upregulated liver glucose and oxidative metabolism assessed by ²H/¹³C flux analysis. VitE supplementation failed to reduce the histological features of NASH. Rather, WD+VitE increased the abundance and saturation of liver ceramides and accelerated metabolic flux dysregulation compared with 8 weeks of WD alone. In summary, VitE did not limit NASH pathogenesis in genetically obese mice, but instead increased some indicators of metabolic dysfunction.

The pregnane X receptor (PXR) is a nuclear receptor that can be activated by numerous drugs and xenobiotic chemicals. PXR thereby functions as a xenobiotic sensor to coordinately regulate host responses to xenobiotics by transcriptionally regulating many genes involved in xenobiotic metabolism. We have previously reported that PXR has pro-atherogenic effects in animal models, but how PXR contributes to atherosclerosis development in different tissues or cell types remains elusive. In this study, we generated an LDL receptor-deficient mouse model with myeloid-specific PXR deficiency (PXR^MyeLDLR^–/–) to elucidate the role of macrophage PXR signaling in atherogenesis. The myeloid PXR deficiency did not affect metabolic phenotypes and plasma lipid profiles, but PXR^MyeLDLR^–/– mice had significantly decreased atherosclerosis at both aortic root and brachiocephalic arteries compared with control littermates. Interestingly, the PXR deletion did not affect macrophage adhesion and migration properties, but reduced lipid accumulation and foam cell formation in the macrophages. PXR deficiency also led to decreased expression of the scavenger receptor CD36 and impaired lipid uptake in macrophages of the PXR^MyeLDLR^–/– mice. Further, RNA-Seq analysis indicated that treatment with a prototypical PXR ligand affects the expression of many atherosclerosis-related genes in macrophages in vitro. These findings reveal a pivotal role of myeloid PXR signaling in atherosclerosis development and suggest that PXR may be a potential therapeutic target in atherosclerosis management.

ABSTRACT

Human genetics influence a range of pathological and clinical phenotypes in respiratory infections; however, the contributions of disease modifiers remain underappreciated. We exploited the Collaborative Cross (CC) mouse genetic-reference population to map genetic modifiers that affect the severity of Pseudomonas aeruginosa lung infection. Screening for P. aeruginosa respiratory infection in a cohort of 39 CC lines exhibits distinct disease phenotypes ranging from complete resistance to lethal disease. Based on major changes in the survival times, a quantitative-trait locus (QTL) was mapped on murine chromosome 3 to the genomic interval of Mb 110.4 to 120.5. Within this locus, composed of 31 protein-coding genes, two candidate genes, namely, dihydropyrimidine dehydrogenase (Dpyd) and sphingosine-1-phosphate receptor 1 (S1pr1), were identified according to the level of genome-wide significance and disease gene prioritization. Functional validation of the S1pr1 gene by pharmacological targeting in C57BL/6NCrl mice confirmed its relevance in P. aeruginosa pathophysiology. However, in a cohort of Canadian patients with cystic fibrosis (CF) disease, regional genetic-association analysis of the syntenic human locus on chromosome 1 (Mb 97.0 to 105.0) identified two single-nucleotide polymorphisms (rs10875080 and rs11582736) annotated to the Dpyd gene that were significantly associated with age at first P. aeruginosa infection. Thus, there is evidence that both genes might be implicated in this disease. Our results demonstrate that the discovery of murine modifier loci may generate information that is relevant to human disease progression.

IMPORTANCE Respiratory infection caused by P. aeruginosa is one of the most critical health burdens worldwide. People affected by P. aeruginosa infection include patients with a weakened immune system, such as those with cystic fibrosis (CF) genetic disease or non-CF bronchiectasis. Disease outcomes range from fatal pneumonia to chronic life-threatening infection and inflammation leading to the progressive deterioration of pulmonary function. The development of these respiratory infections is mediated by multiple causes. However, the genetic factors underlying infection susceptibility are poorly known and difficult to predict. Our study employed novel approaches and improved mouse disease models to identify genetic modifiers that affect the severity of P. aeruginosa lung infection. We identified candidate genes to enhance our understanding of P. aeruginosa infection in humans and provide a proof of concept that could be exploited for other human pathologies mediated by bacterial infection.

ABSTRACT

Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe and fatal acute respiratory disease in humans and remains endemic in the Middle East since first being identified in 2012. There are currently no approved vaccines or therapies available for MERS-CoV. In this study, we evaluated parainfluenza virus 5 (PIV5)-based vaccine expressing the MERS-CoV envelope spike protein (PIV5/MERS-S) in a human DPP4 knockin C57BL/6 congenic mouse model (hDPP4 KI). Following a single-dose intranasal immunization, PIV5-MERS-S induced neutralizing antibody and robust T cell responses in hDPP4 KI mice. A single intranasal administration of 10⁴ PFU PIV5-MERS-S provided complete protection against a lethal challenge with mouse-adapted MERS-CoV (MERS_MA6.1.2) and improved virus clearance in the lung. In comparison, single-dose intramuscular immunization with 10⁶ PFU UV-inactivated MERS_MA6.1.2 mixed with Imject alum provided protection to only 25% of immunized mice. Intriguingly, an influx of eosinophils was observed only in the lungs of mice immunized with inactivated MERS-CoV, suggestive of a hypersensitivity-type response. Overall, our study indicated that PIV5-MERS-S is a promising effective vaccine candidate against MERS-CoV infection.

IMPORTANCE MERS-CoV causes lethal infection in humans, and there is no vaccine. Our work demonstrates that PIV5 is a promising vector for developing a MERS vaccine. Furthermore, success of PIV5-based MERS vaccine can be employed to develop a vaccine for emerging CoVs such as SARS-CoV-2, which causes COVID-19.

ABSTRACT

Obesity and associated metabolic disorders are worldwide public health issues. The gut microbiota plays a key role in the pathophysiology of diet-induced obesity. Glycerol monolaurate (GML) is a widely consumed food emulsifier with antibacterial properties. Here, we explore the anti-obesity effect of GML (1,600 mg/kg of body weight) in high-fat diet (HFD)-fed mice. HFD-fed mice were treated with 1,600 mg/kg GML. Integrated microbiome, metabolome, and transcriptome analyses were used to systematically investigate the metabolic effects of GML, and antibiotic treatment was used to assess the effects of GML on the gut microbiota. Our data indicated that GML significantly reduced body weight and visceral fat deposition, improved hyperlipidemia and hepatic lipid metabolism, and ameliorated glucose homeostasis and inflammation in HFD-fed mice. Importantly, GML modulated HFD-induced gut microbiota dysbiosis and selectively increased the abundance of Bifidobacterium pseudolongum. Antibiotic treatment abolished all the GML-mediated metabolic improvements. A multiomics (microbiome, metabolome, and transcriptome) association study showed that GML significantly modulated glycerophospholipid metabolism, and the abundance of Bifidobacterium pseudolongum strongly correlated with the metabolites and genes that participated in glycerophospholipid metabolism. Our results indicated that GML may be provided for obesity prevention by targeting the gut microbiota and regulating glycerophospholipid metabolism.

Pathway proteomics strategies measure protein expression changes in specific cellular processes that carry out related functions. Using targeted tandem mass tags-based sample multiplexing, hundreds of proteins can be quantified across 10 or more samples simultaneously. To facilitate these highly complex experiments, we introduce a strategy that provides complete control over...

Although pain is a prevalent nonmotor symptom in Parkinson’s disease (PD), it is undertreated, in part because of our limited understanding of the underlying mechanisms. Considering that the basal ganglia are implicated in pain sensation, and that their synaptic outputs are controlled by the subthalamic nucleus (STN), we hypothesized that...

Yi Huang, Jazmin Osorio Mendoza, Catherine Hambly, Baoguo Li, Zengguang Jin, Li Li, Moshen Madizi, Sumei Hu, and John R. Speakman

The heat dissipation limit theory predicts lactating female mice consuming diets with lower specific dynamic action (SDA) should have enhanced lactation performance. Dietary fat has lower SDA than other macronutrients. Here we tested the effects of graded dietary fat levels on lactating Swiss mice. We fed females five diets varying in fat content from 8.3 to 66.6%. Offspring of mothers fed diets of 41.7% fat and above were heavier and fatter at weaning compared to those of 8.3% and 25% fat diets. Mice on dietary fat contents of 41.7% and above had greater metabolizable energy intake at peak lactation (8.3%: 229.4±39.6, 25%: 278.8±25.8, 41.7%: 359.6±51.5, 58.3%: 353.7±43.6, 66.6%: 346±44.7 kJ day^–1), lower daily energy expenditure (8.3%: 128.5±16, 25%: 131.6±8.4, 41.7%: 124.4±10.8, 58.3%: 115.1±10.5, 66.6%: 111.2±11.5 kJ day^–1) and thus delivered more milk energy to their offspring (8.3%: 100.8±27.3, 25%: 147.2±25.1, 41.7%: 225.1±49.6, 58.3%: 238.6±40.1, 66.6%: 234.8±41.1 kJ day^–1). Milk fat content (%) was unrelated to dietary fat content, indicating females on higher fat diets (> 41.7%) produced more rather than richer milk. Mothers consuming diets with 41.7% fat or above enhanced their lactation performance compared to those on 25% or less, probably by diverting dietary fat directly into the milk, thereby avoiding the costs of lipogenesis. At dietary fat contents above 41.7% they were either unable to transfer more dietary fat to the milk, or they chose not to do so, potentially because of a lack of benefit to the offspring that were increasingly fatter as maternal dietary fat increased.

Jing Wen, Tingbei Bo, Xueying Zhang, Zuoxin Wang, and Dehua Wang

Ambient temperature and food composition can affect energy metabolism of the host. Thermal transient receptor potential (thermo-TRPs) ion channels can detect temperature signals and are involved in the regulation of thermogenesis and energy homeostasis. Further, the gut microbiota has also been implicated in thermogenesis and obesity. In the present study, we tested the hypothesis that thermo-TRPs and gut microbiota are involved in reducing diet-induced obesity (DIO) during low temperature exposure. C57BL/6J mice in obese (body mass gain >45%), lean (body mass gain <15%), and control (body mass gain<1%) groups were exposed to high (23±1°C) or low (4±1°C) ambient temperature for 28 days. Our data showed that low temperature exposure attenuated DIO, but enhanced brown adipose tissue (BAT) thermogenesis. Low temperature exposure also resulted in increased norepinephrine (NE) concentrations in the hypothalamus, decreased TRP melastatin 8 (TRPM8) expression in the small intestine, and altered composition and diversity of gut microbiota. In DIO mice, there was a decrease in overall energy intake along with a reduction in TRP ankyrin 1 (TRPA1) expression and an increase in NE concentration in the small intestine. DIO mice also showed increases in Oscillospira, [Ruminococcus], Lactococcus, and Christensenella and decreases in Prevotella, Odoribacter, and Lactobacillus at the genus level in fecal samples. Together, our data suggest that thermos-TRPs and gut microbiota are involved in thermogenesis and energy metabolism during low temperature exposure in DIO mice.

Mutations in retinaldehyde-binding protein 1 (RLBP1), encoding the visual cycle protein cellular retinaldehyde-binding protein (CRALBP), cause an autosomal recessive form of retinal degeneration. By binding to 11-cis-retinoid, CRALBP augments the isomerase activity of retinoid isomerohydrolase RPE65 (RPE65) and facilitates 11-cis-retinol oxidation to 11-cis-retinal. CRALBP also maintains the 11-cis configuration and protects against unwanted retinaldehyde activity. Studying a sibling pair that is compound heterozygous for mutations in RLBP1/CRALBP, here we expand the phenotype of affected individuals, elucidate a previously unreported phenotype in RLBP1/CRALBP carriers, and demonstrate consistencies between the affected individuals and Rlbp1/Cralbp−/− mice. In the RLBP1/CRALBP-affected individuals, nonrecordable rod-specific electroretinogram traces were recovered after prolonged dark adaptation. In ultrawide-field fundus images, we observed radially arranged puncta typical of RLBP1/CRALBP-associated disease. Spectral domain-optical coherence tomography (SD-OCT) revealed hyperreflective aberrations within photoreceptor-associated bands. In short-wavelength fundus autofluorescence (SW-AF) images, speckled hyperautofluorescence and mottling indicated macular involvement. In both the affected individuals and their asymptomatic carrier parents, reduced SW-AF intensities, measured as quantitative fundus autofluorescence (qAF), indicated chronic impairment in 11-cis-retinal availability and provided information on mutation severity. Hypertransmission of the SD-OCT signal into the choroid together with decreased near-infrared autofluorescence (NIR-AF) provided evidence for retinal pigment epithelial cell (RPE) involvement. In Rlbp1/Cralbp−/− mice, reduced 11-cis-retinal levels, qAF and NIR-AF intensities, and photoreceptor loss were consistent with the clinical presentation of the affected siblings. These findings indicate that RLBP1 mutations are associated with progressive disease involving RPE atrophy and photoreceptor cell degeneration. In asymptomatic carriers, qAF disclosed previously undetected visual cycle deficiency.

Motor protein-based active transport is essential for mRNA localization and local translation in animal cells, yet how mRNA granules interact with motor proteins remains poorly understood. Using an unbiased yeast two–hybrid screen for interactions between murine RNA-binding proteins (RBPs) and motor proteins, here we identified protein interaction with APP tail-1 (PAT1) as a potential direct adapter between zipcode-binding protein 1 (ZBP1, a β-actin RBP) and the kinesin-I motor complex. The amino acid sequence of mouse PAT1 is similar to that of the kinesin light chain (KLC), and we found that PAT1 binds to KLC directly. Studying PAT1 in mouse primary hippocampal neuronal cultures from both sexes and using structured illumination microscopic imaging of these neurons, we observed that brain-derived neurotrophic factor (BDNF) enhances co-localization of dendritic ZBP1 and PAT1 within granules that also contain kinesin-I. PAT1 is essential for BDNF-stimulated neuronal growth cone development and dendritic protrusion formation, and we noted that ZBP1 and PAT1 co-locate along with β-actin mRNA in actively transported granules in living neurons. Acute disruption of the PAT1–ZBP1 interaction in neurons with PAT1 siRNA or a dominant-negative ZBP1 construct diminished localization of β-actin mRNA but not of Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) mRNA in dendrites. The aberrant β-actin mRNA localization resulted in abnormal dendritic protrusions and growth cone dynamics. These results suggest a critical role for PAT1 in BDNF-induced β-actin mRNA transport during postnatal development and reveal a new molecular mechanism for mRNA localization in vertebrates.

Virus-like-particle (VLP) influenza vaccines can be given intramuscularly (i.m.) or intranasally (i.n.) and may have advantages over split-virion formulations in the elderly. We tested a plant-made VLP vaccine candidate bearing the viral hemagglutinin (HA) delivered either i.m. or i.n. in young and aged mice. Young adult (5- to 8-week-old) and aged (16- to 20-month-old) female BALB/c mice received a single 3-μg dose based on the HA (A/California/07/2009 H1N1) content of a plant-made H1-VLP (i.m. or i.n.) split-virion vaccine (i.m.) or were left naive. After vaccination, humoral and splenocyte responses were assessed, and some mice were challenged. Both VLP and split vaccines given i.m. protected 100% of the young animals, but the VLP group lost the least weight and had stronger humoral and cellular responses. Compared to split-vaccine recipients, aged animals vaccinated i.m. with VLP were more likely to survive challenge (80% versus 60%). The lung viral load postchallenge was lowest in the VLP i.m. groups. Mice vaccinated with VLP i.n. had little detectable immune response, but survival was significantly increased. In both age groups, i.m. administration of the H1-VLP vaccine elicited more balanced humoral and cellular responses and provided better protection from homologous challenge than the split-virion vaccine.

Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome (RTT). To identify functional pathways that could inform therapeutic entry points, we carried out a genetic screen for secondary mutations that improved phenotypes in Mecp2/Y mice after mutagenesis with N-ethyl-N-nitrosourea (ENU). Here, we report the isolation of 106 founder animals that show suppression of Mecp2-null traits from screening 3177 Mecp2/Y genomes. Whole-exome sequencing, genetic crosses, and association analysis identified 22 candidate genes. Additional lesions in these candidate genes or pathway components associate variant alleles with phenotypic improvement in 30 lines. A network analysis shows that 63% of the genes cluster into the functional categories of transcriptional repression, chromatin modification, or DNA repair, delineating a pathway relationship with MECP2. Many mutations lie in genes that modulate synaptic signaling or lipid homeostasis. Mutations in genes that function in the DNA damage response (DDR) also improve phenotypes in Mecp2/Y mice. Association analysis was successful in resolving combinatorial effects of multiple loci. One line, which carries a suppressor mutation in a gene required for cholesterol synthesis, Sqle, carries a second mutation in retinoblastoma binding protein 8, endonuclease (Rbbp8, also known as CtIP), which regulates a DDR choice in double-stranded break (DSB) repair. Cells from Mecp2/Y mice have increased DSBs, so this finding suggests that the balance between homology-directed repair and nonhomologous end joining is important for neuronal cells. In this and other lines, two suppressor mutations confer greater improvement than one alone, suggesting that combination therapies could be effective in RTT.

Sickle cell disease (SCD) is associated with overactive bladder (OAB). Detrusor overactivity, a component of OAB, is present in an SCD mouse, but the molecular mechanisms for this condition are not well-defined. We hypothesize that nitric oxide (NO)/ ras homolog gene family (Rho) A/Rho-associated kinase (ROCK) dysregulation is a mechanism for detrusor overactivity and that NO-releasing nanoparticles (NO-nps), a novel NO delivery system, may serve to treat this condition. Male adult SCD transgenic, combined endothelial NO synthases (eNOSs) and neuronal NOS (nNOS) gene-deficient (dNOS^–/–), and wild-type (WT) mice were used. Empty nanoparticle or NO-np was injected into the bladder, followed by cystometric studies. The expression levels of phosphorylated eNOS (Ser-1177), protein kinase B (Akt) (Ser-473), nNOS (Ser-1412), and myosin phosphatase target subunit 1 (MYPT1) (Thr-696) were assessed in the bladder. SCD and dNOS^–/– mice had a greater (P < 0.05) number of voiding and nonvoiding contractions compared with WT mice, and they were normalized by NO-np treatment. eNOS (Ser-1177) and AKT (Ser-473) phosphorylation were decreased (P < 0.05) in the bladder of SCD compared with WT mice and reversed by NO-np. Phosphorylated MYPT1, a marker of the RhoA/ROCK pathway, was increased (P < 0.05) in the bladder of SCD mice compared with WT and reversed by NO-np. nNOS phosphorylation on positive (Ser-1412) regulatory site was decreased (P < 0.05) in the bladder of SCD mice compared with WT and was not affected by NO-np. NO-nps did not affect any of the measured parameters in WT mice. In conclusion, dysregulation of NO and RhoA/ROCK pathways is associated with detrusor overactivity in SCD mice; NO-np reverses these molecular derangements in the bladder and decreases detrusor overactivity.

Receptor-mediated endocytosis, which contributes to a wide range of cellular functions, including receptor signaling, cell adhesion, and migration, requires endocytic vesicle release by the large GTPase dynamin 2. Here, the role of dynamin 2 was investigated in platelet hemostatic function using both pharmacological and genetic approaches. Dnm2^fl/fl Pf4-Cre (Dnm2^Plt–/–) mice specifically lacking dynamin 2 within the platelet lineage developed severe thrombocytopenia and bleeding diathesis and Dnm2^Plt–/– platelets adhered poorly to collagen under arterial shear rates. Signaling via the collagen receptor GPVI was impaired in platelets treated with the dynamin GTPase inhibitor dynasore, as evidenced by poor protein tyrosine phosphorylation, including that of the proximal tyrosine kinase Lyn on its activating tyrosine 396 residue. Platelet stimulation via GPVI resulted in a slight decrease in GPVI, which was maintained by dynasore treatment. Dynasore-treated platelets had attenuated function when stimulated via GPVI, as evidenced by reduced GPIbα downregulation, α-granule release, integrin αIIbβ3 activation, and spreading onto immobilized fibrinogen. By contrast, responses to the G-protein coupled receptor agonist thrombin were minimally affected by dynasore treatment. GPVI expression was severely reduced in Dnm2^Plt–/– platelets, which were dysfunctional in response to stimulation via GPVI, and to a lesser extent to thrombin. Dnm2^Plt–/– platelets lacked fibrinogen in their α-granules, but retained von Willebrand factor. Taken together, the data show that dynamin 2 plays a proximal role in signaling via the collagen receptor GPVI and is required for fibrinogen uptake and normal platelet hemostatic function.

MG53 is a member of the TRIM protein family that is predominantly expressed in striated muscles and participates in cell membrane repair. Controversy exists regarding MG53’s role in insulin signaling and manifestation of diabetes. We generated db/db mice with either whole-body ablation or sustained elevation of MG53 in the bloodstream in order to evaluate the physiological function of MG53 in diabetes. To quantify the amount of MG53 protein in circulation, we developed a monoclonal antibody against MG53 with high specificity. Western blot using this antibody revealed lower or no change of serum MG53 levels in db/db mice or patients with diabetes compared with control subjects. Neither whole-body ablation of MG53 nor sustained elevation of MG53 in circulation altered insulin signaling and glucose handling in db/db mice. Instead, mice with ablation of MG53 were more susceptible to streptozotocin-induced dysfunctional handling of glucose compared with the wild-type littermates. Alkaline-induced corneal injury demonstrated delayed healing in db/db mice, which was restored by topical administration of recombinant human (rh)MG53. Daily intravenous administration of rhMG53 in rats at concentrations up to 10 mg/kg did not produce adverse effects on glucose handling. These findings challenge the hypothetical function of MG53 as a causative factor for the development of diabetes. Our data suggest that rhMG53 is a potentially safe and effective biologic to treat diabetic oculopathy in rodents.

Diabetic keratopathy occurs in ~70% of all people with diabetes. This study was designed to examine the effects of vitamin D receptor knockout (VDR^–/–) and vitamin D deficiency (VDD) on corneal epithelial wound healing and nerve density in diabetic mice. Diabetes was induced using the low-dose streptozotocin method. Corneal epithelial wounds were created using an Algerbrush, and wound healing was monitored over time. Corneal nerve density was measured in unwounded mice. VDR^–/– and VDD diabetic mice (diabetic for 8 and 20 weeks, respectively) had slower healing ratios than wild-type diabetic mice. VDR^–/– and VDD diabetic mice also showed significantly decreased nerve density. Reduced wound healing ratios and nerve densities were not fully rescued by a supplemental diet rich in calcium, lactose, and phosphate. We conclude that VDR^–/– and VDD significantly reduce both corneal epithelial wound healing and nerve density in diabetic mice. Because the supplemental diet did not rescue wound healing or nerve density, these effects are likely not specifically related to hypocalcemia. This work supports the hypothesis that low vitamin D levels can exacerbate preexisting ophthalmic conditions, such as diabetes.

As an alternative to lifelong insulin supplementation, potentiation of immune tolerance in patients with type 1 diabetes could prevent the autoimmune destruction of pancreatic islet β-cells. This study was aimed to assess whether the G3c monoclonal antibody (mAb), which triggers the glucocorticoid-induced TNFR-related (Gitr) costimulatory receptor, promotes the expansion of regulatory T cells (Tregs) in SV129 (wild-type) and diabetic-prone NOD mice. The delivery of the G3c mAb via G3C hybridoma cells enveloped in alginate-based microcapsules (G3C/cps) for 3 weeks induced Foxp3⁺ Treg-cell expansion in the spleen of wild-type mice but not in Gitr^–/– mice. G3C/cps also induced the expansion of nonconventional Cd4⁺Cd25^–/lowFoxp3^lowGitr^int/high (GITR single-positive [sp]) Tregs. Both Cd4⁺Cd25⁺Gitr^highFoxp3⁺ and GITRsp Tregs (including also antigen-specific cells) were expanded in the spleen and pancreas of G3C/cps-treated NOD mice, and the number of intact islets was higher in G3C/cps-treated than in empty cps-treated and untreated animals. Consequently, all but two G3C/cps-treated mice did not develop diabetes and all but one survived until the end of the 24-week study. In conclusion, long-term Gitr triggering induces Treg expansion, thereby delaying/preventing diabetes development in NOD mice. This therapeutic approach may have promising clinical potential for the treatment of inflammatory and autoimmune diseases.

Inadequate insulin secretion in response to glucose is an important factor for β-cell failure in type 2 diabetes (T2D). Although HMG-CoA reductase degradation 1 (HRD1), a subunit of the endoplasmic reticulum–associated degradation complex, plays a pivotal role in β-cell function, HRD1 elevation in a diabetic setting contributes to β-cell dysfunction. We report in this study the excessive HRD1 expression in islets from humans with T2D and T2D mice. Functional studies reveal that β-cell–specific HRD1 overexpression triggers impaired insulin secretion that will ultimately lead to severe hyperglycemia; by contrast, HRD1 knockdown improves glucose control and response in diabetic models. Proteomic analysis results reveal a large HRD1 interactome, which includes v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), a master regulator of genes implicated in the maintenance of β-cell function. Furthermore, mechanistic assay results indicate that HRD1 is a novel E3 ubiquitin ligase that targets MafA for ubiquitination and degradation in diabetic β-cells, resulting in cytoplasmic accumulation of MafA and in the reduction of its biological function in the nucleus. Our results not only reveal the pathological importance of excessive HRD1 in β-cell dysfunction but also establish the therapeutic importance of targeting HRD1 in order to prevent MafA loss and suppress the development of T2D.

Vitamin D deficiency has been associated with increased incidence of diabetes, both in humans and in animal models. In addition, an association between vitamin D receptor (VDR) gene polymorphisms and diabetes has also been described. However, the involvement of VDR in the development of diabetes, specifically in pancreatic β-cells, has not been elucidated yet. Here, we aimed to study the role of VDR in β-cells in the pathophysiology of diabetes. Our results indicate that Vdr expression was modulated by glucose in healthy islets and decreased in islets from both type 1 diabetes and type 2 diabetes mouse models. In addition, transgenic mice overexpressing VDR in β-cells were protected against streptozotocin-induced diabetes and presented a preserved β-cell mass and a reduction in islet inflammation. Altogether, these results suggest that sustained VDR levels in β-cells may preserve β-cell mass and β-cell function and protect against diabetes.

Yan Xing, Hongling Wei, Xiumei Xiao, Zekun Chen, Hui Liu, Xiaomei Tong, and Wei Zhou

Infants with intrauterine growth retardation (IUGR) have a high risk of developing bronchial asthma in childhood, but the underlying mechanisms remain unclear. This study aimed to disclose the role of vascular non-inflammatory molecule 1 (vannin-1, encoded by the Vnn1 gene) and its downstream signaling in IUGR asthmatic mice induced by ovalbumin. Significant histological alterations and an increase of vannin-1 expression were revealed in IUGR asthmatic mice, accompanied by elevated methylation of Vnn1 promoter regions. In IUGR asthmatic mice, we also found (i) a direct binding of HNF4α and PGC1α to Vnn1 promoter by ChIP assay; (ii) a direct interaction of HNF4α with PGC1α; (iii) upregulation of phospho-PI3K p85/p55 and phospho-Akt^Ser473 and downregulation of phospho-PTENT^yr366, and (iv) an increase in nuclear NFB p65 and a decrease in cytosolic IB-α. In primary cultured bronchial epithelial cells derived from the IUGR asthmatic mice, knockdown of Vnn1 prevented upregulation of phospho-Akt^Ser473 and an increase of reactive oxygen species (ROS) and TGF-β production. Taken together, we demonstrate that elevated vannin-1 activates the PI3K/Akt/NFB signaling pathway, leading to ROS and inflammation reactions responsible for asthma occurrence in IUGR individuals. We also disclose that interaction of PGC1α and HNF4α promotes methylation of Vnn1 promoter regions and then upregulates vannin-1 expression.

This study showed that treatment with a therapeutic monoclonal immunoglobulin-G1 antibody against phosphorylcholine on oxidized phospholipids preserves coronary flow reserve and attenuates atherosclerotic inflammation as determined by the uptake of ¹⁸F-fluorodeoxyglucose in atherosclerotic mice. The noninvasive imaging techniques represent translational tools to assess the efficacy of phosphorylcholine-targeted therapy on coronary artery function and atherosclerosis in clinical studies.

Of four genotypes of Encephalitozoon cuniculi, E. cuniculi genotype II is considered to represent a parasite that occurs in many host species in a latent asymptomatic form, whereas E. cuniculi genotype III seems to be more aggressive, and infections caused by this strain can lead to the death of even immunocompetent hosts. Although albendazole has been considered suitable for treatment of Encephalitozoon species, its failure in control of E. cuniculi genotype III infection has been reported. This study determined the effect of a 100x recommended daily dose of albendazole on an Encephalitozoon cuniculi genotype III course of infection in immunocompetent and immunodeficient mice and compared the results with those from experiments performed with a lower dose of albendazole and E. cuniculi genotype II. The administration of the regular dose of abendazole during the acute phase of infection reduced the number of affected organs in all strains of mice and absolute counts of spores in screened organs. However, the effect on genotype III was minor. Surprisingly, no substantial effect was recorded after the use of a 100x dose of albendazole, with larger reductions seen only in the number of affected organs and absolute counts of spores in all strains of mice, implying variations in albendazole resistance between these Encephalitozoon cuniculi genotypes. These results imply that differences in the course of infection and the response to treatment depend not only on the immunological status of the host but also on the genotype causing the infection. Understanding how microsporidia survive in hosts despite targeted antimicrosporidial treatment could significantly contribute to research related to human health.

Subtractive immunization is useful for directing the immune response away from dominant epitopes to allow antibodies against less immunogenic antigens to be developed. It is also useful when there is a need to downplay the response to carriers added to peptides and recombinant proteins, such as Gst and Ig, to aid in production and purification. Subtractive immunization protocols also can be helpful if the target protein is part of a mixture (such as a cell lysate) or present on the surface of transfected cells.

The repetitive immunization at multiple sites (RIMMS) protocol capitalizes on the animal's innate immune system, which is genetically preprogrammed to recognize many antigens. By repetitively immunizing the animal, B cells that recognize the antigen are kept continuously expanding until the lymph nodes are harvested for hybridoma generation. This is a good method for making a more diverse repertoire of antibodies or antibodies directed against conformational epitopes.

Researchers at Washington University School of Medicine in St. Louis found that gene therapy in mice helped build strength and significant muscle mass quickly, while reducing the severity of osteoarthritis. The gene therapy also prevented obesity, even when the mice were fed a high-fat diet.

Obese mice with unhealthy lifestyles gain significantly less weight and avoid type 2 diabetes when they receive bacteriophages from the faeces of a lean mouse, according to a new University of Copenhagen study.