Mouse Peripheral Tissues Research Articles

Glucose metabolism controls monocyte homeostasis and migration but has no impact on atherosclerosis development in mice

Monocytes directly contribute to atherosclerosis development by their recruitment to plaques in which they differentiate into macrophages. In the present study, we ask how modulating monocyte glucose metabolism could affect their homeostasis and their impact on atherosclerosis. Here we investigate how circulating metabolites control monocyte behavior in blood, bone marrow and peripheral tissues of mice. We find that serum glucose concentrations correlate with monocyte numbers. In diet-restricted mice, monocytes fail to metabolically reprogram from glycolysis to fatty acid oxidation, leading to reduced monocyte numbers in the blood. Mechanistically, Glut1-dependent glucose metabolism helps maintain CD115 membrane expression on monocytes and their progenitors, and regulates monocyte migratory capacity by modulating CCR2 expression. Results from genetic models and pharmacological inhibitors further depict the relative contribution of different metabolic pathways to the regulation of CD115 and CCR2 expression. Meanwhile, Glut1 inhibition does not impact atherosclerotic plaque development in mouse models despite dramatically reducing blood monocyte numbers, potentially due to the remaining monocytes having increased migratory capacity. Together, these data emphasize the role of glucose uptake and intracellular glucose metabolism in controlling monocyte homeostasis and functions.

Human NMO-IgG Induced Different Pathological and Immunological Changes in the CNS and Peripheral Tissues of Mice.

The majority of neuromyelitis optica spectrum disorders (NMOSD) patients are seropositive for aquaporin-4 (AQP4)-specific antibodies [also named neuromyelitis optica immunoglobulin G antibodies (NMO-IgG)]. Although NMO-IgG can induce pathological changes in the central nervous system (CNS), the immunological changes in the CNS and peripheral tissue remain largely unknown. We investigated whether NMO-IgG binds to tissue expressing AQP4 and induces immunological changes in the peripheral tissue and CNS. C57BL/6 female mice were assigned into an NMOSD or control group. Pathological and immunological changes in peripheral tissue and CNS were measured by immunostaining and flow cytometry, respectively. Motor impairment was measured by open-field test. We found that NMO-IgG did bind to astrocyte- and AQP4-expressing peripheral tissue, but induced glial fibrillary acidic protein and AQP4 loss only in the CNS. NMO-IgG induced the activation of microglia and modulated microglia polarization toward the classical (M1) phenotype, but did not affect innate or adaptive immune cells in the peripheral immune system, such as macrophages, neutrophils, Th17/Th1, or IL-10-producing B cells. In addition, NMOSD mice showed significantly less total distance traveled and higher immobility time in the open field. We found that injection of human NMO-IgG led to astrocytopathic lesions with microglial activation in the CNS. However, there were no significant pathological or immunological changes in the peripheral tissues.

Automated Capillary Electrophoresis Immunoblot for the Detection of Alpha-Synuclein in Mouse Tissue.

Alpha-synuclein (aSyn) is a key player in neurodegenerative diseases such as Parkinson's disease (PD), dementia with Lewy bodies, or multiple system atrophy. aSyn is expressed throughout the brain, and can also be detected in various peripheral tissues. In fact, initial symptoms of PD are non-motoric and include autonomic dysfunction, suggesting that the periphery might play an important role in early development of the disease. aSyn is expressed at relatively low levels in non-central tissues, which brings challenges for its detection and quantification in different tissues. Our goal was to assess the sensitivity of aSyn detection in central and peripheral mouse tissues through capillary electrophoresis (CE) immunoblot, considering the traditional SDS-PAGE immunoblot as the current standard. Tissues from central and non-central origin from wild type mice were extracted, and included midbrain, inner ear, and esophagus/stomach. aSyn detection was assessed through immunoblotting using Simple Western size-based CE and SDS-PAGE. CE immunoblots show a consistent detection of aSyn in central and peripheral tissues. Through SDS-PAGE, immunoblots revealed a reliable signal corresponding to aSyn, particularly following membrane fixation. Our results suggest a reliable detection of aSyn in central and peripheral tissues using the CE Simple Western immunoblot system. These observations can serve as preliminary datasets when aiming to formally compare CE with SDS-PAGE, as well as for further characterization of aSyn using this technique.

Inhibiting arachidonic acid generation mitigates aging-induced hyperinsulinemia and insulin resistance in mice

BackgroundAging-related type 2 diabetes (T2DM) is characterized by hyperinsulinemia, insulin resistance, and β-cell dysfunction. However, the underlying molecular mechanisms remain to be unclear. MethodsWe conducted non-targeted metabolomics to compare human serum samples from young adults (YA), elderly adults (EA), and elderly adults with diabetes (EA+DM) of Chinese population. Adult mice and aged mice were intragastrically administered with varespladib every day for two weeks and metabolic characteristics were monitored. Serum levels of arachidonic acid, insulin, and C-peptide, as well as serum activity of secretory phospholipase A2 (sPLA2) were detected in mice. Mouse islet perfusion assays were used to assess insulin secretion ability. Phosphorylated AKT levels were measured to evaluate insulin sensitivities of peripheral tissues in mice. ResultsNon-targeted metabolomics analysis of human serum samples revealed differential metabolic signatures among the YA, EA, and EA+DM groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed significant enhancement of arachidonic acid metabolism and glycerophospholipid metabolism in the EA group compared with the YA group. Further analysis identified two metabolic fluxes that favored the accumulation of arachidonic acid in the elderly. Increased levels of arachidonic acid were also confirmed in aged mice with hyperinsulinemia and insulin resistance, together with subsequent glucose intolerance. Conversely, inhibiting the generation of arachidonic acid with varespladib, an inhibitor of sPLA2, reduced aging-associated diabetes by improving hyperinsulinemia and hepatic insulin resistance in aged mice but not in adult mice. Islet perfusion assays also showed that varespladib treatment suppressed the enhanced insulin secretion observed in aged islets. ConclusionsCollectively, our findings uncover that arachidonic acid serves as a metabolic hub in Chinese elderly population. Our results also suggest that arachidonic acid plays a fundamental role in regulating β-cell function during aging and point to a novel therapy for aging-associated diabetes.

GFRAL Is Widely Distributed in the Brain and Peripheral Tissues of Mice.

In 2017, four independent publications described the glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as receptor for the growth differentiation factor 15 (GDF15, also MIC-1, NAG-1) with an expression exclusively in the mice brainstem area postrema (AP) and nucleus tractus solitarii (NTS) where it mediates effects of GDF15 on reduction of food intake and body weight. GDF15 is a cell stress cytokine with a widespread expression and pleiotropic effects, which both seem to be in contrast to the reported highly specialized localization of its receptor. This discrepancy prompts us to re-evaluate the expression pattern of GFRAL in the brain and peripheral tissues of mice. In this detailed immunohistochemical study, we provide evidence for a more widespread distribution of this receptor. Apart from the AP/NTS region, GFRAL-immunoreactivity was found in the prefrontal cortex, hippocampus, nucleus arcuatus and peripheral tissues including liver, small intestine, fat, kidney and muscle tissues. This widespread receptor expression, not taken into consideration so far, may explain the multiple effects of GDF-15 that are not yet assigned to GFRAL. Furthermore, our results could be relevant for the development of novel pharmacological therapies for physical and mental disorders related to body image and food intake, such as eating disorders, cachexia and obesity.

Nesfatin-1 regulates steroidogenesis in mouse Leydig cells

Nesfatin-1 is a polypeptide hormone known to regulate appetite and energy metabolism and is derived from the precursor protein nucleobindin 2 (NUCB2). Recent studies have shown that nesfatin-1 is expressed in many peripheral tissues in mice, including the reproductive organs. However, its function and regulation in the testis remain unknown. In this study, we investigated the expression of Nucb2 mRNA and nesfatin-1 protein in mouse Leydig cells and the Leydig cell line, TM3 cells. We also examined whether Nucb2 mRNA expression is regulated by gonadotropins and whether exogenous nesfatin-1 affects steroidogenesis in primary Leydig cells isolated from the testis and TM3 cells. We found that Nucb2 mRNA and nesfatin-1 protein were present in primary Leydig cells and TM3 cells, and nesfatin-1 binding sites were also found in both cell types. Nucb2 mRNA expression in testis, primary Leydig cells, and TM3 cells was increased after treatment with pregnant mare’s serum gonadotropin and human chorionic gonadotropin. After nesfatin-1 treatment, the expression of steroidogenesis-related enzyme genes Cyp17a1 and Hsd3b was upregulated in primary Leydig cells and TM3 cells. Our results suggest that NUCB2/nesfatin-1 expression in mouse Leydig cells may be regulated through the hypothalamic-pituitary-gonadal axis and that nesfatin-1 produced by Leydig cells may locally regulate steroidogenesis in an autocrine manner. This study provides insight into the regulation of NUCB2/nesfatin-1 expression in Leydig cells and the effect of nesfatin-1 on steroidogenesis, which may have implications for male reproductive health.

Local and systemic administration of AAV vectors with alphaherpesvirus latency-associated promoter 2 drives potent transgene expression in mouse liver, kidney, and skeletal muscle

Adeno-associated virus (AAV) has great potential as a source of treatments for conditions that might respond to potent and ubiquitous transgene expression. However, among its drawbacks, the genetic “payload” of AAV vectors is limited to <4.9 kb and some commonly used gene promoters are sizeable and susceptible to transcriptional silencing. We recently described a short (404 bp), potent, and persistent promoter obtained from the genome of pseudorabies virus (PrV) called alphaherpesvirus latency-associated promoter 2 (LAP2). Here, we evaluated the biodistribution and potency of transgene expression in mouse peripheral tissues in response to local and systemic administration of AAV8-LAP2 and AAV9-LAP2. We found that administration of these vectors resulted in levels of transgene expression that were similar to the larger EF1α promoter. LAP2 drives potent transgene expression in mouse liver and kidney when administered systemically and in skeletal muscle in response to intramuscular delivery. Notably, in skeletal muscle, administration of vectors with LAP2 and EF1α promoters resulted in preferential transduction of myofibers type 2. A direct side-by-side comparison between LAP2 and the EF1α promoter revealed that, despite its smaller size, LAP2 was equally potent to the EF1α promoter and resulted in widespread gene expression after IV and IM administration of AAV8 or AAV9 vectors. Collectively, these findings suggest that constructs that include LAP2 may have the capacity to deliver large therapeutically effective payloads in support of future gene therapy protocols.

Protein Transduction Domain-Mediated Delivery of Recombinant Proteins and In Vitro Transcribed mRNAs for Protein Replacement Therapy of Human Severe Genetic Mitochondrial Disorders: The Case of Sco2 Deficiency

Mitochondrial disorders represent a heterogeneous group of genetic disorders with variations in severity and clinical outcomes, mostly characterized by respiratory chain dysfunction and abnormal mitochondrial function. More specifically, mutations in the human SCO2 gene, encoding the mitochondrial inner membrane Sco2 cytochrome c oxidase (COX) assembly protein, have been implicated in the mitochondrial disorder fatal infantile cardioencephalomyopathy with COX deficiency. Since an effective treatment is still missing, a protein replacement therapy (PRT) was explored using protein transduction domain (PTD) technology. Therefore, the human recombinant full-length mitochondrial protein Sco2, fused to TAT peptide (a common PTD), was produced (fusion Sco2 protein) and successfully transduced into fibroblasts derived from a SCO2/COX-deficient patient. This PRT contributed to effective COX assembly and partial recovery of COX activity. In mice, radiolabeled fusion Sco2 protein was biodistributed in the peripheral tissues of mice and successfully delivered into their mitochondria. Complementary to that, an mRNA-based therapeutic approach has been more recently considered as an innovative treatment option. In particular, a patented, novel PTD-mediated IVT-mRNA delivery platform was developed and applied in recent research efforts. PTD-IVT-mRNA of full-length SCO2 was successfully transduced into the fibroblasts derived from a SCO2/COX-deficient patient, translated in host ribosomes into a nascent chain of human Sco2, imported into mitochondria, and processed to the mature protein. Consequently, the recovery of reduced COX activity was achieved, thus suggesting the potential of this mRNA-based technology for clinical translation as a PRT for metabolic/genetic disorders. In this review, such research efforts will be comprehensibly presented and discussed to elaborate their potential in clinical application and therapeutic usefulness.

L-type Amino Acid Transporter 1 Utilizing Ferulic Acid Derivatives Show Increased Drug Delivery in the Mouse Pancreas Along with Decreased Lipid Peroxidation and Prostaglandin Production.

Oxidative stress and pathological changes of Alzheimer's disease (AD) overlap with metabolic diseases, such as diabetes mellitus (DM). Therefore, tackling oxidative stress with antioxidants is a compelling drug target against multiple chronic diseases simultaneously. Ferulic acid (FA), a natural antioxidant, has previously been studied as a therapeutic agent against both AD and DM. However, FA suffers from poor bioavailability and delivery. As a solution, we have previously reported about L-type amino acid transporter 1 (LAT1)-utilizing derivatives with increased brain delivery and efficacy. In the present study, we evaluated the pharmacokinetics and antioxidative efficacy of the two derivatives in peripheral mouse tissues. Furthermore, we quantified the LAT1 expression in studied tissues with a targeted proteomics method to verify the transporter expression in mouse tissues. Additionally, the safety of the derivatives was assessed by exploring their effects on hemostasis in human plasma, erythrocytes, and endothelial cells. We found that both derivatives accumulated substantially in the pancreas, with over a 100-times higher area under curve compared to the FA. Supporting the pharmacokinetics, the LAT1 was highly expressed in the mouse pancreas. Treating mice with the LAT1-utilizing derivative of FA lowered malondialdehyde and prostaglandin E2 production in the pancreas, highlighting its antioxidative efficacy. Additionally, the LAT1-utilizing derivatives were found to be hemocompatible in human plasma and endothelial cells. Since antioxidative derivative 1 was substantially delivered into the pancreas along the previously studied brain, the derivative can be considered as a safe dual-targeting drug candidate in both the pancreas and the brain.

Skipping breakfast regimen induces an increase in body weight and a decrease in muscle weight with a shifted circadian rhythm in peripheral tissues of mice.

Meal timing is a key factor in synchronising the circadian clock in peripheral tissues. Circadian disorders are associated with the metabolic syndrome. Previously, we demonstrated that a skipping breakfast regimen (SBR) with a high-fat diet increased body weight gain in rats. In this study, we investigated whether SBR with a normal diet led to abnormal lipid metabolism and muscle metabolism in mice. Male C57BL/6 mice were fed during zeitgeber time (ZT) 12-24 in the control group and ZT 16-24 in the SBR group for 2 weeks. SBR mice showed increased body weight gain and perirenal adipose tissue weight. The plantar muscle weight was decreased in the SBR group compared with that in the control group. Furthermore, SBR delayed the circadian oscillations in clock gene expression in peripheral tissues, such as the liver, adipose tissue and muscle, as well as the oscillations in the expression of lipid metabolism-related genes in the liver and adipose tissue. These results suggest that skipping breakfast over a long period of time is associated with a risk of obesity, the metabolic syndrome and muscle loss, such as sarcopenia.

Protein Profiling of RGS6, a Pleiotropic Gene Implicated in Numerous Neuropsychiatric Disorders, Reveals Multi-Isoformic Expression and a Novel Brain-Specific Isoform.

A metanalysis identified regulator of G-protein signaling 6 (RGS6) as one of 23 loci with pleiotropic effects on four or more human psychiatric disorders. This finding is significant as it confirms/extends the findings of numerous other studies implicating RGS6 in CNS function and pathology. RGS6 is a highly conserved member of the RGS protein family whose cellular roles are likely affected by mRNA splicing and alternative domain inclusion/exclusion. Indeed, we previously identified multiple RGS6 splice variants predicted to produce 36 distinct protein isoforms containing either long (RGS6L) or short (RGS6S) N-terminal domains, an incomplete or intact GGL domain, and nine alternative C termini. Unfortunately, sequence similarities between the isoforms have made it difficult to confirm their individual existence and/or to determine their unique functions. Here, we developed three RGS6-specific antibodies that recognize all RGS6 protein isoforms (RGS6-fl), the N-terminus of RGS6L isoforms (RGS6-L), and an 18-amino acid alternate C-terminal sequence (RGS6-18). Using these antibodies, we demonstrate that RGS6L(+GGL) isoforms, predominating in both mouse (both sexes) CNS and peripheral tissues, are most highly expressed in the CNS. We further identify three novel RGS6 protein bands that are larger (61, 65, and 69-kDa) than the ubiquitously expressed 53- to 57-kDa RGS6L(+GGL) proteins. Importantly, we show that the 69-kDa protein is a brain-specific dephospho form of the 65-kDa band, the first identified phosphorylated RGS6 isoform. Together, these data begin to define the functional significance behind the complexity of RGS6 gene processing and further clarifies RGS6’s physiological roles by resolving tissue-specific RGS6 protein expression.

Biodistribution of Adeno-Associated Virus Serotype 5 Viral Vectors Following Intrathecal Injection.

The pharmacokinetic profile of AAV particles following intrathecal delivery has not yet been clearly defined. The present study evaluated the distribution profile of adeno-associated virus serotype 5 (AAV5) viral vectors following lumbar intrathecal injection in mice. After a single bolus intrathecal injection, viral DNA concentrations in mouse whole blood, spinal cord, and peripheral tissues were determined using quantitative polymerase chain reaction (qPCR). The kinetics of AAV5 vector in whole blood and the concentration over time in spinal and peripheral tissues were analyzed. Distribution of the AAV5 vector to all levels of the spinal cord, dorsal root ganglia, and into systemic circulation occurred rapidly within 30 min following injection. Vector concentration in whole blood reached a maximum 6 h postinjection with a half-life of approximately 12 h. Area under the curve data revealed the highest concentration of vector distributed to dorsal root ganglia tissue. Immunohistochemical analysis revealed AAV5 particle colocalization with the pia mater at the spinal cord and macrophages in the dorsal root ganglia (DRG) 30 min after injection. These results demonstrate the widespread distribution of AAV5 particles through cerebrospinal fluid and preferential targeting of DRG tissue with possible clearance mechanisms via DRG macrophages.

A multi-tissue multi-omics analysis reveals distinct kineztics in entrainment of diurnal transcriptomes by inverted feeding

SummaryTime of eating synchronizes circadian rhythms of metabolism and physiology. Inverted feeding can uncouple peripheral circadian clocks from the central clock located in the suprachiasmatic nucleus. However, system-wide changes of circadian metabolism and physiology entrained to inverted feeding in peripheral tissues remain largely unexplored. Here, we performed a 24-h global profiling of transcripts and metabolites in mouse peripheral tissues to study the transition kinetics during inverted feeding, and revealed distinct kinetics in phase entrainment of diurnal transcriptomes by inverted feeding, which graded from fat tissue (near-completely entrained), liver, kidney, to heart. Phase kinetics of tissue clocks tracked with those of transcriptomes and were gated by light-related cues. Integrated analysis of transcripts and metabolites demonstrated that fatty acid oxidation entrained completely to inverted feeding in heart despite the slow kinetics/resistance of the heart clock to entrainment by feeding. This multi-omics resource defines circadian signatures of inverted feeding in peripheral tissues (www.CircaMetDB.org.cn).

A Multi-Tissue Multi-Omics Analysis Reveals Distinct Kinetics in Entrainment of Diurnal Transcriptomes by Inverted Feeding

Time of eating synchronizes circadian rhythms of metabolism and physiology. Inverted feeding reverses peripheral circadian clocks, but privileges the light-responsive central clock located in the suprachiasmatic nucleus. However, system-wide changes of circadian metabolism and physiology entrained to inverted feeding in peripheral tissues remain largely unexplored. Here, we performed a 24-h global profiling of transcripts and metabolites in mouse peripheral tissues to study the transition kinetics during inverted feeding, and revealed distinct kinetics in phase entrainment of diurnal transcriptomes by inverted feeding, which graded from fat tissue (near-completely entrained), liver, kidney, to heart. Phase kinetics of tissue clocks tracked with those of transcriptomes, and was gated by light cue. Integrated analysis of transcripts and metabolites demonstrated that fatty acid oxidation entrained completely to inverted feeding in heart despite the slow kinetics of the heart clock. This multi-omics resource defines circadian signatures of inverted feeding in peripheral tissues (www.CircaMetDB.org.cn).

CNS Transduction Benefits of AAV-PHP.eB over AAV9 Are Dependent on Administration Route and Mouse Strain

Adeno-associated viral (AAV) vectors are attractive tools for central nervous system (CNS) gene therapy because some vectors can cross the blood-brain barrier (BBB), allowing them to be used as minimally invasive treatments. A novel AAV vector recently evolved in vivo, AAV-PHP.eB, has been reported to cross the BBB more effectively than the existing gold standard AAV9, but not under all conditions. Here, we compared the efficacy of single-stranded AAV-PHP.eB and AAV9 in targeting mouse CNS and peripheral tissues after administration via various routes, in two different mouse strains (C57BL/6J and B6C3), and after packaging AAV-PHP.eB with a self-complementary genome. We found that AAV-PHP.eB produced higher CNS transduction than AAV9 after intravenous injection, but only in C57BL/6J and not in B6C3 mice. AAV-PHP.eB and AAV9 produced similar CNS transduction when the administration route did not require the vectors to cross the BBB. Packaging AAV-PHP.eB with a self-complementary genome increased overall CNS transduction, but at the expense of strong neuronal tropism. AAV-PHP.eB resulted in less transduction of liver tissue than AAV9 under all conditions. Taken together, these results suggest the potential for AAV-PHP.eB as a vector for CNS gene therapy applications, but consideration will be required for translation beyond mouse models.

Differential expression of receptors mediating receptor-mediated transcytosis (RMT) in brain microvessels, brain parenchyma and peripheral tissues of the mouse and the human

Receptor-mediated transcytosis (RMT) is a principal pathway for transport of macromolecules essential for brain function across the blood–brain barrier (BBB). Antibodies or peptide ligands which bind RMT receptors are often co-opted for brain delivery of biotherapeutics. Constitutively recycling transferrin receptor (TfR) is a prototype receptor utilized to shuttle therapeutic cargos across the BBB. Several other BBB-expressed receptors have been shown to mediate transcytosis of antibodies or protein ligands including insulin receptor (INSR) and insulin-like growth factor-1 receptor (IGF1R), lipid transporters LRP1, LDLR, LRP8 and TMEM30A, solute carrier family transporter SLC3A2/CD98hc and leptin receptor (LEPR). In this study, we analyzed expression patterns of genes encoding RMT receptors in isolated brain microvessels, brain parenchyma and peripheral organs of the mouse and the human using RNA-seq approach. IGF1R, INSR and LRP8 were highly enriched in mouse brain microvessels compared to peripheral tissues. In human brain microvessels only INSR was enriched compared to either the brain or the lung. The expression levels of SLC2A1, LRP1, IGF1R, LRP8 and TFRC were significantly higher in the mouse compared to human brain microvessels. The protein expression of these receptors analyzed by Western blot and immunofluorescent staining of the brain microvessels correlated with their transcript abundance. This study provides a molecular transcriptomics map of key RMT receptors in mouse and human brain microvessels and peripheral tissues, important to translational studies of biodistribution, efficacy and safety of antibodies developed against these receptors.

Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Is Highly Expressed in Mouse Tissues With Metabolic Function.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) form a family of atypical growth factors discovered for their neuroprotective properties in the central nervous system (CNS) in animal models of neurodegenerative diseases. Although their mechanism of protective action still remains unclear, it has been suggested that both MANF and CDNF promote cell survival through regulating the unfolded protein response (UPR), thereby relieving endoplasmic reticulum (ER) stress. Recent studies identified MANF for its emerging roles in metabolic function, inflammation and pancreatic β-cells. We have found that MANF deletion from the pancreas and β-cells leads to postnatal depletion of β-cells and diabetes. Moreover, global MANF-deficiency in mice results in severe diabetes-independent growth retardation. As the expression pattern of MANF in mouse tissues has not been extensively studied, we set out to thoroughly investigate MANF expression in embryonic and adult mice using immunohistochemistry, histochemical X-gal staining, enzyme-linked immunosorbent assay (ELISA), and quantitative reverse transcription PCR (RT-qPCR). We found that MANF is highly expressed in brain neurons regulating energy homeostasis and appetite, as well as in hypothalamic nuclei producing hormones and neuropeptides important for different body functions. Strong expression of MANF was also observed in peripheral mouse tissues and cells with high secretory and metabolic function. These include pituitary gland and interestingly we found that the anterior pituitary gland is smaller in MANF-deficient mice compared to wild-type mice. Consequently, we found reduction in the number of growth hormone- and prolactin-producing cells. This combined with increased expression of UPR genes, reduced number of proliferating cells in the anterior pituitary and dysregulated expression of pituitary hormones might contribute to the severe growth defect seen in the MANF knockout mice. Moreover, in this study we compared MANF and CDNF levels in mouse tissues. Unlike MANF, CDNF protein levels are generally lower in mouse tissues, and the highest levels of CDNF was observed in the tissues with high-energy demands and oxidative roles, including heart, muscle, testis, and brown adipose tissue.

Pharmacokinetics of HLDF-6-AA Peptide in the Organism of Experimental Animals

Pharmacokinetics of the promising antitumor peptide HLDF-6-AA (Ac-ThrGlyGluAsnHisArg-NH2) was studied using its uniformly tritiated derivative. Experiments were performed on male Wistar rats, Balb/c mice and Chinchilla rabbits. The tritium labeled peptide [3H]HLDF-6-AA with the molar radioactivity of 50 Ci/mmol was obtained by the reaction of high-temperature solid-state catalytic isotope exchange (HSCIE). Under intravenous bolus administration of HLDF-6-AA peptide to rats and rabbits, the characteristics of its pharmacokinetic profile in the blood were obtained and the values of the main pharmacokinetic parameters of HLDF-6-AA peptide, and its active metabolite HisArg-NH2 were calculated. It was shown that parameters of the retention time in the body and the rate of elimination for peptides HLDF-6-AA and HisArg-NH2 in rats and rabbits are close and in rats they are about 7 and 21 min, respectively. It was shown that repeated administration of the drug does not lead to a change in its regular cumulation and does not cause a change in its pharmacokinetics compared with a single administration. The linearity of the dependence of pharmacokinetic parameters on the amount of administered peptide in the range of 2–22 mg/kg was proved in experiments in rats. As a result of the study of the distribution of HLDF-6-AA peptide and its metabolite HisArg-NH2 between the blood and peripheral tissues of mice, it was shown that the maximum concentration of HLDF-6-AA peptide is observed in the kidney tissues and a somewhat smaller concentration in the omentum. It was found that 15–20 min after intraperitoneal administration of HLDF-6-AA to mice, the concentration of HisArg-NH2 peptide begins to exceed the concentration of HLDF-6-AA peptide, which is caused by its greater resistance to proteolytic hydrolysis. The highest concentration of HisArg-NH2 peptide is observed in the kidney and liver tissues.

Targeted invalidation of SR-B1 in macrophages reduces macrophage apoptosis and accelerates atherosclerosis.

SR-B1 is a cholesterol transporter that exerts anti-atherogenic properties in liver and peripheral tissues in mice. Bone marrow (BM) transfer studies suggested an atheroprotective role in cells of haematopoietic origin. Here, we addressed the specific contribution of SR-B1 in the monocyte/macrophage. We generated mice deficient for SR-B1 in monocytes/macrophages (Lysm-Cre × SR-B1f/f) and transplanted their BM into Ldlr-/- mice. Fed a cholesterol-rich diet, these mice displayed accelerated aortic atherosclerosis characterized by larger macrophage-rich areas and decreased macrophage apoptosis compared with SR-B1f/f transplanted controls. These findings were reproduced in BM transfer studies using another atherogenic mouse recipient (SR-B1 KOliver × Cholesteryl Ester Transfer Protein). Haematopoietic reconstitution with SR-B1-/- BM conducted in parallel generated similar results to those obtained with Lysm-Cre × SR-B1f/f BM; thus suggesting that among haematopoietic-derived cells, SR-B1 exerts its atheroprotective role primarily in monocytes/macrophages. Consistent with our in vivo data, free cholesterol (FC)-induced apoptosis of macrophages was diminished in the absence of SR-B1. This effect could not be attributed to differential cellular cholesterol loading. However, we observed that expression of apoptosis inhibitor of macrophage (AIM) was induced in SR-B1-deficient macrophages, and notably upon FC-loading. Furthermore, we demonstrated that macrophages were protected from FC-induced apoptosis by AIM. Finally, AIM protein was found more present within the macrophage-rich area of the atherosclerotic lesions of SR-B1-deficient macrophages than controls. Our findings suggest that macrophage SR-B1 plays a role in plaque growth by controlling macrophage apoptosis in an AIM-dependent manner.

Age- and gender-dependent D-amino acid oxidase activity in mouse brain and peripheral tissues: implication for aging and neurodegeneration.

D-amino acid oxidase (DAO) is a flavoenzyme, catalysing oxidative deamination of D-amino acids to produce corresponding α-keto acids, ammonia and hydrogen peroxide. In our search for DAO activity among various tissues, we developed a sensitive assay based on hydrogen peroxide production involving enzyme-coupled colorimetric assay with peroxidase. We first optimized buffer components to extract DAO protein from mouse tissues. Here we show that DAO activity was detected in kidney, cerebellum, medulla oblongata, midbrain and spinal cord, but not in liver. In addition, we observed that DAO activity and expression were decreased in thoracic and lumbar regions of spinal cord in aged mice when compared with young mice, indicating that decreased DAO is involved in motoneuron degeneration during senescence. We also found gender difference in DAO activity in the kidney, suggesting that DAO activity is influenced by sexual dimorphism. We newly detected DAO activity in the epididymis, although undetected in testis. Furthermore, DAO activity was significantly higher in the caput region than corpus and cauda regions of epididymis, indicating that D-amino acids present in the testis are eliminated in epididymis. Taken together, age- and gender-dependent DAO activity in each organ may underlie the human pathophysiology regulated by D-amino acid metabolism.