Secreted proprotein convertase subtilisin/kexin type 9 reduces both hepatic and extrahepatic low-density lipoprotein receptors in vivo

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease that is known to reduce hepatic low-density lipoprotein receptor (LDLR) levels and increase plasma LDL cholesterol. It is not clear, however, whether secreted PCSK9 degrades extrahepatic LDLRs. We present evidence that recombinant PCSK9, either injected intravenously into or expressed in the liver of C57BL/6 mice, significantly reduced LDLR levels in multiple extrahepatic tissues. During the initial characterization, we found that injected human recombinant PCSK9 at 30 μg/mouse had a half-life of 15 min in serum in mice. Hepatic LDLR levels were reduced within 30 min and the degradation of hepatic LDLR reached the maximum 2 h after the initial protein injection. Endocytosis of PCSK9 in liver occurred within 5 min of protein injection and internalized PCSK9 was only barely detectable within 1 h. When extrahepatic LDLRs were examined by Western blotting analysis, we found significant reductions of LDLRs in multiple extrahepatic tissues including lung, adipose and kidney along with the more dramatic reduction of LDLRs in liver. These studies were further extended using adenoviral expression of human PCSK9 in C57BL/6 mice to demonstrate that PCSK9 produced in liver impacted extrahepatic tissue LDLR levels as well. Taken together, our studies indicate that secreted PCSK9 can potentially impact extrahepatic tissue cholesterol homeostasis by regulating extrahepatic tissue LDLR levels.     

An anti-ANGPTL3/8 antibody decreases circulating triglycerides by binding to a LPL-inhibitory leucine zipper-like motif

Triglycerides (TG) are required for fatty acid transport and storage and are essential for human health. Angiopoietin-like-protein 8 (ANGPTL8) has previously been shown to form a complex with ANGPTL3 that increases circulating TG by potently inhibiting LPL. We also recently showed that the TG-lowering apolipoprotein A5 (ApoA5) decreases TG levels by suppressing ANGPTL3/8-mediated LPL inhibition. To understand how LPL binds ANGPTL3/8 and ApoA5 blocks this interaction, we used hydrogen-deuterium exchange mass-spectrometry and molecular modeling to map binding sites of LPL and ApoA5 on ANGPTL3/8. Remarkably, we found that LPL and ApoA5 both bound a unique ANGPTL3/8 epitope consisting of N-terminal regions of ANGPTL3 and ANGPTL8 that are unmasked upon formation of the ANGPTL3/8 complex. We further used ANGPTL3/8 as an immunogen to develop an antibody targeting this same epitope. After refocusing on antibodies that bound ANGPTL3/8, as opposed to ANGPTL3 or ANGPTL8 alone, we utilized bio-layer interferometry to select an antibody exhibiting high-affinity binding to the desired epitope. We revealed an ANGPTL3/8 leucine zipper-like motif within the anti-ANGPTL3/8 epitope, the LPL-inhibitory region, and the ApoA5-interacting region, suggesting the mechanism by which ApoA5 lowers TG is via competition with LPL for the same ANGPTL3/8-binding site. Supporting this hypothesis, we demonstrate that the anti-ANGPTL3/8 antibody potently blocked ANGPTL3/8-mediated LPL inhibition in vitro and dramatically lowered TG levels in vivo. Together, these data show that an anti-ANGPTL3/8 antibody targeting the same leucine zipper-containing epitope recognized by LPL and ApoA5 markedly decreases TG by suppressing ANGPTL3/8-mediated LPL inhibition.     

White‐tailed deer exploit temporal refuge from multi‐predator and human risks on roads

Although most prey have multiple predator species, few studies have quantified how prey respond to the temporal niches of multiple predators which pose different levels of danger. For example, intraspecific variation in diel activity allows white‐tailed deer (Odocoileus virginianus) to reduce fawn activity overlap with coyotes (Canis latrans) but finding safe times of day may be more difficult for fawns in a multi‐predator context. We hypothesized that within a multi‐predator system, deer would allocate antipredation behavior optimally based on combined mortality risk from multiple sources, which would vary depending on fawn presence. We measured cause‐specific mortality of 777 adult (>1‐year‐old) and juvenile (1–4‐month‐old) deer and used 300 remote cameras to estimate the activity of deer, humans, and predators including American black bears (Ursus americanus), bobcats (Lynx rufus), coyotes, and wolves (Canis lupus). Predation and vehicle collisions accounted for 5.3 times greater mortality in juveniles (16% mortality from bears, coyotes, bobcats, wolves, and vehicles) compared with adults (3% mortality from coyotes, wolves, and vehicles). Deer nursery groups (i.e., ≥1 fawn present) were more diurnal than adult deer without fawns, causing fawns to have 24–38% less overlap with carnivores and 39% greater overlap with humans. Supporting our hypothesis, deer nursery groups appeared to optimize diel activity to minimize combined mortality risk. Temporal refuge for fawns was likely the result of carnivores avoiding humans, simplifying diel risk of five species into a trade‐off between diurnal humans and nocturnal carnivores. Functional redundancy among multiple predators with shared behaviors may partially explain why white‐tailed deer fawn predation rates are often similar among single‐ and multi‐predator systems.     

Vergleichende Untersuchungen über die Wirkung von Korrelationsstörung und geotropischer Reizung bei Gramineenkoleoptilen

Ohne ZusammenfassungMit 7 Textabbildungen.     

Testosterone, androstenedione, and androstenediol: effects on the initiation of mating behavior of inexperienced castrated male rats

The effectiveness of testosterone, androstenediol, and androstenedione in initiating sexual behavior in inexperienced castrated male rats was studied. Androgens were injected daily for 32 days at three dose levels (0.3, 1, and 3 mg) for each androgen. Among the three effective androgens tested, testosterone was found to be the most potent one in initiating copulatory behavior while androstenedione was the least potent one.     

Localization and distribution of gap junctions in normal and cardiomyopathic hamster heart

Gap junctions in mammalian heart function to provide low-resistance channels between adjacent cells for passage of ions and small molecules. It is clear that the almost unrestricted passage of ions between cells, ionic coupling, is required for coordinate and synchronous contraction. This knowledge of gap junction function has made it important to study their properties in normal and abnormal tissues. In the present study, we analyzed gap junction distribution in normal and cardiomyopathic heart tissue utilizing immunofluorescent and electron microscopy techniques. Frozen, unfixed sections of age-matched normal and cardiomyopathic cardiac tissues were immunofiuorescently stained using an antibody directed against a specific peptide sequence of the connexin-43 gap junction protein. These studies revealed a characteristic punctate staining pattern for the intercalated discs in normal tissues. Some of the intercalated discs in cardiomyopathic hearts appeared to stain normally; however, others stained diffusely. The pixel intensity distribution of the confocal images demonstrated a marked difference of up to 90% increase in the number of pixels in cardiomyopathic myocardium (CM), yet the pixel intensity of gap junctions had a decrease of approximately 60%. This suggests the possibility that connexin-43 is present in CM cells in significant quantity; however, it does not become localized on the membranes as in normal cells. Electron-microscopic findings corroborate these observations on CM cells by showing an irregular distribution of intercalated discs relatively smaller in size with abnormal orientation and distribution. © 1994 Wiley-Liss, Inc.     

Sex differences in the regulation of embryonic brain aromatase

Oestrogen formed from androgen by aromatization plays a critical role in the sexual differentiation of the male brain and behaviour. A question which has still to be answered is what regulates the gender-specific changes in aromatase activity forming oestrogen during sensitive periods of brain growth. Using a primary cell culture technique and sexed embryos, we have shown that in the fetal mouse brain, oestrogen formation in the male is neuronal rather than glial and aromatase activity is regionally localized, being higher in the hypothalamus than in the cortex. The aromatase activity measured from cells in culture has the same enzyme binding affinity (apparent K_m 40 nM) as intact brain samples. Neurones developing in the embryonic male brain (embryonic day (ED) 15) contain higher aromatase activity (V_max, 895 fmol/h/mg protein) than the female (V_max, 604). Although a sex difference exists at early stages of embryonic development (ED 13), the embryonic aromatase system is regulated by steroids later in fetal development. The developing aromatase-containing neuroblasts probably form processes which connect to other aromatase neurones. Immunoreactive staining with an aromatase polyclonal antibody identifies an increase in numbers of aromatase-immunoreactive hypothalamic neuronal cell bodies following testosterone treatment. Testosterone treatment also causes both stimulation of neurite growth and branching as well as functional maturation of aromatase neurones. In particular, there is an increase in aromatase activity per neurone as well as a dramatic increase in the number of neurones expressing the enzyme. Both the functional and morphological changes depend on androgen receptor stimulation for several days in vitro. This conclusion is supported by colocalization studies which reveal a high number of fetal hypothalamic aromatase neurones co-expressing androgen receptor. We conclude that testosterone influences the growth of male hypothalamic neurones containing aromatase at a sensitive period of brain development. Endogenous steroid inhibitors of aromatase, probably formed within the neuroglia, also play a role in the control of oestrogen production. An endogenous 5-reduced metabolite of testosterone, 5-androstanedione, is almost as potent in inhibiting neuronal hypothalamic aromatase activity (K_i = 23 nM) as the synthetic non-steroidal inhibitors such as the imidazole, fadrozole, and the triazoles, arimidex and letrozole. It is clear that the oestrogen-forming capacity of the male hypothalamus has the special characteristics and plasticity of regulation which could affect brain differentiation at specific steroid-sensitive stages in ontogeny.